The Animal Core is the center’s scientific hub for catalyzing research on in vivo genetic analysis and synergism between alcohol and risk factors for the pathogenesis of alcoholic liver and pancreatic diseases (ALPD) by investigators within and outside of the center.
One prime feature of the Core is its ability to provide different versions of the mouse intragastric (iG) and ad lib ethanol feeding models which produce clinically relevant pathologic spectra of ALPD resulting from alcohol and second or multiple hits commonly associated with ALPD patients. To promote investigation on synergism between alcohol and obesity, the Core has developed a new mouse hybrid model of ad lib feeding of Western diet and iG ethanol infusion which produces alcoholic liver fibrosis and the hybrid plus binge model which, for the first time, reproduces histological evidence of neutrophilic alcoholic hepatitis. Alcohol-fed HCV NS5A/Core transgenic (Tg) models produce liver tumor and are successfully utilized for isolation and characterization of tumor-initiating cells. Using knockout, knock-in, and Tg mice, these models allow invaluable genetic deletion and addition analysis, providing researchers the unique opportunity to test the importance of a single gene of interest in the genesis and evolution of ALPD. The Core also incorporates chimeric and genetic mice into the iG models to test the role of bone marrow-derived cells and to enable genetic tracing of macrophages, hepatic progenitor cells, hepatic stellate cells, liver mesothelial cells in the evolution of alcoholic liver injury, inflammation, fibrosis and cancer.
The Core currently has the capacity to produce ~750 rodent models per year, of which ~65 percent are served as the iG model. From 2015 to 2020, the Core has produced 4783 rodents for 38 center members and 15 non-center scientists and contributed to 102 publications by center members and four publications by non-center investigators, 50 grants acquired by center members and 23 grants acquired/applied by 16 non-center investigators. The Core also facilitated 46 collaborative projects via the shared use of animals and tissues, and application of complementary scientific expertise to the models. With the innovative models and techniques, the Core strives to serve the ALPD scientific community as a unique and invaluable national resource.
Models Provided by the Animal Core
Different Versions of Rodent iG Models
Mouse Intragastric Ethanol Infusion Model (Mouse iG-Std)
The iG-Std model is prepared and provided by the Animal Core of the Southern California Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis supported by the National Institute on Alcohol Abuse and Alcoholism (P50AA11999). 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 mortality rate for the mouse iG-Std model for 4 wk feeding is 6.3+1.2%. These mortalities usually result from inadvertent technical errors, aggressive animals destroying the catheters, accidental over-intoxication or withdrawal.
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. Ueno A et al. Mouse intragastric infusion (iG) model. Nat. Protoc. 7:771-81, 2012
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
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:
- 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.
- 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]
Hybrid HCFD+Alc+Binge Model
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 2nd 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]
Composite physiologic and pathologic data of these different versions of the iG model produced by the Core in 2014, are summarized below.
|Model||Regimen||Duration||Alcohol Dose||BAL (mg%)||ALT (U/L)||Histology|
|iG-std||HFD/alcohol iG||1-8 wk||~33g/kg||309+51||269+29||ASH|
|iG-OF||170% Cal HFD/alcohol||8 wk||~33g/kg||285+28||392+28||Severe ASH|
|Hybrid||HCFD ad lib/alcohol iG||8-12 wk||~27g/kg||434+92||398+38||ASH with liver fibrosis|
|Hybrid + Binge||Hybrid/weekly binge||8-12 wk||~27g/kg
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.
Ad lib HCFD+Alc or Ad lib HCFD+Alc+Binge
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
Prioritization, Scheduling Requests, and Chargebacks
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 (firstname.lastname@example.org) 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.
|CENTER MEMBER PRICING**|
|Standard iG||Hybrid iG||Lieber-DeCarli|
|NON-CENTER MEMBER PRICING|
|Standard iG||Hybrid iG||Lieber-DeCarli|
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.
|Type||Duration||Chargeback (Mouse)||Chargeback (Rat)|
|Bile Duct Ligation (BDL) – induced liver fibrosis||1~3 weeks||$200~$300 per mouse||$250~$450 per rat|
|CCl4 (sc) – induced liver fibrosis||4~8 weeks||$150~$300 per mouse||$250~$600 per rat|
|Porcine serum (ip) -induced liver fibrosis||6~8 weeks||N/A||$250~$450 per rat|
|Thioacetamide (in water)-induced liver fibrosis||8~12 weeks||$300~$450 per mouse||$600~$800 per rat|
|Partial hepatectomy (PHx)||~1 wk||$200 per mouse||$250 per rat|
|DEN-initiated Western Alcohol Diet (WAD)-promoted liver tumor||5~6 months||$500~$600 per mouse||N/A|
|Liver tumor initiating cells (TIC) orthotopic transplantation||$200 per mouse||N/A|
- 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.
Animal Core Tissue Sharing Program
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. Total 18 investigators have already benefited from the program during the current funding cycle by receiving liver, pancreas and intestinal samples from the mouse iG model. Tissue sharing is facilitated by contacting the Animal Core personnel for pending experiments to be conducted by the Animal Core.
Routine BAL and ALT measurements
The Core offers services of measuring blood alcohol levels (BAL) on a chargeback basis. We offer these services for two main reasons: firstly, the Core performs this assay with a small required volume (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, tryglycerides besides alcohol, again requiring only 10μl per assay. Secondly, we believe it important to standardize the assay technique for this key parameter when we standardize the models to be provided to the investigators. Thus, the Core’s standardized BAL assay enables comparisons of the results on alcohol intoxication among the investigators using the standardized models. The assay’s 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 alcoholic rats. Our inter-assay variability for alcohol assessed from the most recent 20 assays are 2.4+0.7%.
Terminal experiments and sample collection in the Core’s laboratory
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, those PIs whose laboratories are not located on the USC campus can send laboratory personnel on the date(s) of the completion of the Core’s services for the terminal procedures. If PIs cannot send personnel, the Core personnel can perform the routine terminal procedures on a charge-back basis ($35-$40/hr including supply costs). The Core also arranges the samples to be sent to the PIs’ laboratories in dry ice packages via air courier. Our routine procedure includes: 1) general anesthesia with ketamine and xylazine unless contraindicated; 2) weighing body weight; 3) collection of venous blood from inferior vena cava; 4) collection of whole liver or/and pancreas which will be quickly placed into PBS at 4oC; 5) weighing liver and pancreas; 6) Cryo-tissue preservation: tissue slices for fixation in 3% paraformladehyde (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 billiary 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 fat and intestine can be collected according to the investigators’ instructions.
TRAINING AND TISSUE SHARING PROGRAM
The Animal Core facilitates the training of any lab personnel from other institutions on techniques required for setting up a rat and mouse intragastric infusion model.
It also facilitates a tissue sharing program that allows the sharing of tissue specimens obtained from animal models produced by the Core and used by Center investigators.
For more information, please contact:
Dr. Hide Tsukamoto
USC, MMR 414
Tel: (323) 442-5107 or (323) 442-3121
Fax: (323) 442-3126