Animal Model and Cell Isolation Core

The purpose of the Animal Model and Cell Isolation Core is to provide centralized facilities and standardized protocols for in vivo models of acute and chronic ethanol exposure to rodents, as well as the use of in vitro primary cell cultures isolated from ethanol-exposed animals.

We also provide access to our animal/cell models and biorepository as a national and international resource. 


Animal Model Core (back to top)

The major goal of the Animal Models and Cell Isolation Core will be to make available to members of NOAC tissue and cellular samples from control and ethanol-exposed animals.  The availability of centralized facilities will allow rapid access of investigators in NOAC, as well as investigators new to alcohol research, to the tissues and cells needed to test novel and innovative hypotheses without the delay of each PI developing these techniques in each of their own laboratories. 


 
Ad libitum feeding – Lieber DeCarli Ethanol Diet:  (back to top)

A major advantage of ad libitum feeding of ethanol is that it is not technically demanding, generally requiring only daily changes of the ethanol-containing liquid.  In the Lieber-DeCarli control diet, 18% of energy is derived from protein, 35% from fat and 47% from carbohydrate.  In ethanol-containing diets, ethanol provides up to 36% of dietary energy.  Food consumption is monitored daily and body weight 3 times per week.  The ethanol-fed group is allowed free access to ethanol containing diet with increasing concentrations of ethanol. For mice the model is: 1% (vol/vol) (5.5 % of kcal) and 2% (11% of kcal) each for 2 days, then 4% ethanol for 1 wk (22% of kcal), 5% ethanol (27% of kcal) for 1 wk and finally 6% (32% of kcal) ethanol for 1-6 wks (1). For rats the model is: 3.2% (vol/vol) for 2 days, and then 6.4% (vol/vol) for 1-8 wks.  Control animals are pair-fed diets which iso-calorically substitute maltose dextrins for ethanol over the entire feeding period.

Acute Ethanol: 
Mice and rats are exposed to ethanol acutely by gavage. 20% ethanol solutions are prepared from 95% ethanol in sterile water.  Isocaloric dextrin and/or isovolemic saline will be provided to control animals.  Animals are exposed to 1-6 g/kg ethanol.  Peak serum ethanol concentrations are measured after 30-60 min.  Acute ethanol-induced liver injury can be observed 12 h after acute exposure.

Chronic Ethanol: 

Mice: 8-10 week old female or male mice (wild-type and genetically modified, as required for individual investigator’s protocols) are housed in shoe-box cages (2 animals/cage) with microisolator lids. Mice adapt well to the ethanol during this gradual increase in exposure and continue to grow on the Lieber-DeCarli ethanol diet (2).

Rats:  Male Wistar rats are purchased from Harlan Sprague Dawley (Indianapolis, IN).  Rats (150-160 g at initiation of feeding protocol) are allowed ad libitum access to the Lieber-DeCarli ethanol diet or pair-fed a control diet, as previously described (1,3).  Rats are housed in individual wire-bottom cages.  Rats adapt well to this regime and continue to grow on the Lieber-DeCarli ethanol diet.

Moderate Ethanol and CCl4: 
We have established acute and chronic mouse models to evaluate the response to moderate ethanol consumption and CCl4 exposure (4,5). In each model, mice are 10-12 wks old at the start of the studies.  Mice are fed a control-liquid diet for 2 days, then 1% (vol/vol) ethanol diet for 2 days, then 2% (vol/vol) (11% calories) ethanol diet. Animals receive an intraperitoneal (IP) injection of olive oil or CCl4 (prediluted1:3 in olive oil) on day 3 of the 2% ethanol diet. For the acute model, mice receive one dose of 1 µl/g body weight Olive Oil or CCl4. For the chronic model, mice are ramped up to the full dose of CCl4 over two doses (first injection at 0.25 µl/g body weight, the second at 0.5 µl/g body weight). Mice are anesthetized and euthanized at precise time points after the last CCl4 injection.




Ad libitum feeding and second ‘hit’:  (back to top)

The lack of disease progression in ad libitum models of acute alcohol exposure may actually be relevant to human exposure.  Specifically, only a fraction of chronic alcohol drinkers ever develop alcoholic hepatitis, and possibly progress to fibrosis/cirrhosis (6,5). It is thought that a second 'hit' is required to drive liver injury beyond simple steatosis.  Experimentally, the 2nd 'hit' can be an inducer of inflammation (e.g., LPS), or even a binge dose of ethanol itself.

LPS as 2nd hit:
After chronic ethanol feeding, 8-10 wk old mice are challenged with 0.7 μg LPS/g body weight or an equivalent volume of sterile, endotoxin-free saline (0.09%).  Mice are anesthetized and euthanized at precise times after the LPS challenge to monitor acute inflammatory responses to activation of TLR4 (e.g. MyD88 dependent at 1 h, TRIF dependent at 4 h), depending on the experimental question.

Acute ethanol as a 2nd 'hit’:
Here we employ a model in which acute binge alcohol is administered in addition to chronic ad libitum exposure (variously called acute on chronic, Gao-binge, or NIAAA model).  Under these conditions, hepatic injury and inflammation is far more severe; indeed, the additional pathologic changes are similar to mild alcoholic hepatitis in humans. 
8-10 wk old female and male mice are provided ad lib access to ethanol- or pair-fed diets up to 5% ethanol for 10 days.  At that time, mice are then exposed to an acute dose of ethanol at 5g/kg or isocaloric volume of maltose as a vehicle control.  Mice are then euthanized over 0-24 h later and tissues collected.




High-fat diet induced obesity:  (back to top)

Male or female (5-6 wks old) wild type and genetically modified mice (as required for individual investigator’s protocols) will be fed a high fat high sucrose diet (from Research Diets ranging in fat from 45-58% of calories as fat, 26-39% carbohydrate (sucrose) and 16% protein or control diets (10% fat, 73% carbohydrate, 16.4% protein or rodent chow) for up to 16 weeks. Mice will be weighed weekly.




Drug interventions/treatments:  (back to top)

Animal/Cell Core personnel have experience in the acute and chronic treatment of mice and rats with drug interventions.  Personnel have experience in gavage, intraperitoneal injection, intravenous injection, as well as implantation of Alzet mini-osmotic pumps, for long term drug interventions. The Animal/Cell Core will thus be able to provide drug interventions during acute and/or chronic ethanol exposure as required to test specific hypotheses by members of NOAC.




Bone Marrow Transplant:  (back to top)
Donor Mice:  To isolate bone marrow, donor mice (female, 6-12 weeks of age) will be euthanized, and femurs will be dissected free of tissue and flushed with PBS.   Bone marrow cells will be centrifuged and resuspended in PBS.
Recipient mice: 5 week old female mice will be lethally irradiated. Four hours later, mice will receive bone marrow cells in PBS by intravenous injection into the tail vein.
Four weeks after irradiation, mice will be treated with a single dose of clodronate containing liposomes that are diluted in sterile saline and injected intravenously into the tail vein.
These liposomes deplete sessile Kupffer cells that are radio-resistant, and are replenished within 7 days after clodronate.




Cell Isolation Core (back to top)

Hepatocytes, Kupffer cells and hepatic stellate cells are routinely isolated by Animal/Cell Core personnel making use of in situ perfusion protocols (7-11) in both rats and mice.  Cells will be released to individual investigators after the first media change.  Animal/Cell Core is not responsible for specific experimental manipulations.  Isolated hepatocytes, Kupffer cells or stellate cells can also be provided without plating for flow cytometry analysis, as required.  Cell viability is monitored by trypan blue exclusion and purity of the preparations is routinely assessed by morphology, as well as specific staining (ED2 (rat) or F4/80 (mouse)) by flow cytometry or fluorescence microscopy.




Tissue Harvesting (back to top)

The Animal Core routinely provides liver samples to individual investigators either on ice , fixed in formalin for histology, frozen in optimal cutting temperature (OCT) compound for immunohistochemistry, flash frozen or freeze-clamped in liquid nitrogen for biochemical assays or stored in RNA later. Samples are stored appropriately (-80, -20 or 4°C) until further analysis.  Blood is processed to serum or plasma, as required, aliquoted, inventoried and stored appropriately until further analysis.  Additional tissue samples (specific adipose depots, skeletal or cardiac muscle, kidney, intestine, brain/brain regions etc.) are also sampled for individual investigators.  Fixation of tissues via thoracotomy is also available to investigators.




Additional Services (back to top)

Phenotypic characterization of ethanol exposure: 
The Animal/Cell Core will also provide basic phenotyping measures including ALT/AST, triglyceride concentrations, Oil Red O, Sirius red staining, hydroxyproline concentration, blood ethanol and CYP2E1 expression or activity on a fee for service basis for all NOAC investigators.

Zebra fish facility: 
In collaboration with Takuya Sakaguchi, Assistant Staff in Stem Cell Biology at CCF, the NOAC will also provide access to zebra fish exposed to ethanol.  Dr. Sakaguchi is an active member of NOAC and is involved in collaborations with a number of NOAC investigators, which will facilitate incorporation of this useful model organism into our on-going investigations.

Metabolic Phenotyping:
The Mouse Metabolic Phenotyping Center (MMPC) at Case Western Reserve University specializes in hyperinsulinemic euglycemic glucose clamps, glucose tolerance tests, total energy expenditure at rest and exercise, lipid or collagen turnover studies utilizing the incorporation of 2H from 2H-enriched body water, feeding studies, acute or chronic vascular catheterization, activity monitoring, etc.  Dr. Croniger, the director of the MMPC, will consult with NOAC investigators in the design and implementation of a variety of specialized metabolic studies in mice and rats.  Additional details can be found at http://www.case.edu/med/mmpc




Biorepository (back to top)
The Animal Core also maintains an extensive biorepository of mouse and rat tissue/blood samples on experiments conducted since 2006.  Requests for tissues can be made through the Animal Core Manager –Megan McMullen.


Mouse Models Stored in the Biorepository:

Lieber DeCarli Ethanol Liquid Diet

Acute EtOH

Chronic Ethanol Various Times

Binge Ethanol

Acute on Chronic

Acute CCl4 and EtOH

Chronic CCl4 and EtOH

Acute CCl4 Various Times

Chronic CCl4

High Fat Diet Various Times

Controls

WT (C57BL6J)

X

x

x

x

x

x

x

x

x

WT129S1

x

Alb cre

x

x

x

FABP cre

x

x

x

Hif flox

x

x

x

Complement

C1q

x

x

x

x

C4

x

x

x

x

C3aR

x

x

x

C5aR

x

x

CD55

x

x

FD

x

x

x

x

x

x

C3

x

x

x

C1q/FD

x

x

MBL/C1q

x

x

MBL/FD

x

x

Cell Death

RIP3

x

x

x

x

MLK3

x

x

x

Caspase 1

x

x

x

Bid

x

x

x

Mincle

x

Hypoxia

Hepatocyte Specific Hif

x

x

x

x

x

LysM Specific Hif

x

x

FABP Specific HiF

x

x

Soluble IgM

x

Oxidative Stress

Cyp2e1

x

x

x

TRX

x

x

x

x

COX1

x

COX2

x

MPO

x

Innate Immune Response

TNFR1

x

x

MCP1

x

TLR4

x

x

x

MIF

x

x

x

x

x

x

x

NOD

x

IRF3 KO

x

x

IRF3 KI

x

x

IRAK M

x

IRAK  R

x

Other

Rag1

x

x

x

PAF

x

Gli2

x

TRVP4

x

Egr-1

x

x

x

x

A2bR

x

x


Rat Samples Stored in Biorepository:

Lieber DeCarli Ethanol Liquid Diet

Acute  Ethanol

Chronic Ethanol

4 days

1 week

2 weeks

3 weeks

4 weeks

6 weeks

Wistar Rats

x

x

x

x

x

x


Available Tissues:

Mouse:

Rat:

Liver

Liver

Adipose (gonadal/subcutaneous)

Adipose (gonadal)

Kidney

Kidney

Intestine

Plasma

Brain

Lung

Plasma





Request Service (back to top)

To request Animal/Cell Core services, each user will submit a form describing the name of the project, PI and proposed study design. The experimental design will be reviewed by the Core Director – Dr. Colleen Croniger and Manager – Megan McMullen and then scheduled appropriately into the Animal Models and Cell Isolation Core calendar. 

Individual PIs will supply the animals necessary for their experiments to the Core and cover the per diem housing costs for the animals. Users will be charged for diet, drugs and sample collection materials based on the study design. 

If the User requests isolated cells, Users will provide an account number for a charge-back of $100 for each hepatocyte/Kupffer cell isolation, to cover the cost of biochemicals, gradients and cell culture media required for isolation of purified cell populations. 

If there are conflicting/competing requests for access to services, priority will be given to Research Components and Pilot Projects directly supported by the P50.  Next priority will be to NIAAA funded NOAC members and last priority to investigators outside of the NOAC.




LITERATURE CITED

  1. Aldred A and Nagy LE. Ethanol dissociates hormone-stimulated cAMP production from inhibition of TNFa production in rat Kupffer cells. Am J Physiol  276: G98-G106, 1999.

  2. Pritchard MT, McMullen MR, Stavitsky AB, Cohen JI, Lin F, Medof ME, and Nagy LE. Differential contributions of C3, C5, and decay-accelerating factor to ethanol-induced fatty liver in mice. Gastroenterology 132: 1117-26, 2007.

  3. Thakur V, Pritchard MT, McMullen MR, and Nagy LE. Adiponectin normalizes LPS-stimulated TNF-alpha production by rat Kupffer cells after chronic ethanol feeding. Am J Physiol Gastrointest Liver Physiol 290: G998-1007, 2006.

  4. Chiang DJ, Roychowdhury S, Bush K, McMullen MR, Pisano S, Niese K, Olman MA, Pritchard MT, and Nagy LE. Adenosine 2A Receptor Antagonist Prevented and Reversed Liver Fibrosis in a Mouse Model of Ethanol-Exacerbated Liver Fibrosis. PLoS One 8: e69114, 2013.

  5. Roychowdhury S, Chiang DJ, McMullen MR, and Nagy LE. Moderate, chronic ethanol feeding exacerbates carbon-tetrachloride-induced hepatic fibrosis via hepatocyte-specific hypoxia inducible factor 1alpha. Pharmacol Res Perspect 2: e00061, 2014.

  6. Roychowdhury S, Chiang DJ, Mandal P, McMullen MR, Liu X, Cohen JI, Pollard J, Feldstein AE, and Nagy LE.  Inhibition of apoptosis protects mice from ethanol-mediated acceleration of earl markers of CCl4-induced fibrosis but not steatosis or inflammation. Alcohol Clin Exp Res 36 : 1139-47, 2012.

  7. Bakhautdin B, Das D, Mandal P, Roychowdhury S, Danner J, Bush K, Pollard K, Kaspar JW, Li W, Salomon RG, McMullen MR, and Nagy LE. Protective role of HO-1 and carbon monoxide in ethanol-induced hepatocyte cell death and liver injury in mice. J Hepatol 61: 1029-37, 2014.

  8. Kishore R, McMullen MR, and Nagy LE. Stabilization of TNFa mRNA by chronic ethanol: role of A+U rich elements and p38 mitogen activated protein kinase signaling pathway. J Biol Chem 276: 41930-41937, 2001.

  9. Nagy LE. Ethanol metabolism and inhibition of nucleoside uptake lead to increased extracellular adenosine in hepatocytes. Am. J. Physiol. 262: C1175-1180, 1992.

  10. Nagy LE and deSilva SEF. Ethanol increases receptor-dependent cAMP production in cultured hepatocytes by decreasing Gi-mediated inhibition. 286: 681-686, 1992.