3.5 Alcohol Feeding Models
Undoubtedly alcohol consumption increases the risk of acute and recurrent acute pancreatitis and it is thesingle most frequent cause for chronic pancreatitis in humans（Yadav and Lowenfels 2013；Aghdassi et al.2015）.Although there is no clear threshold of an alcohol amount that inevitably leads to pancreatic fibrosis heavy alcohol consumption is associated with a higher risk of developing chronic pancreatitis（Frulloni et al.2009；Irving et al.2009）.The harmful effects of alcohol have been explored in several studies using whole animal or ex-vivo models and there are plausible data on the pathophysiologic way in which ethanol injures the pancreas（Gukovskaya et al.2006；Pfutzer et al.2002）.Alcohol is metabolized either in an oxidative pathway by the successive action of alcohol-dehydrogenases（ADHs）and aldehyde-dehydrogenases（ALDHs）into its metabolites acetaldehyde and acetate.In the non-oxidative pathway ethanol is combined with fatty acids to fatty ethyl esters（FAEEs）（Criddle 2015）.Both pathways occur in the pancreas and both ethanol and its catabolic endproducts are toxic to pancreatic cells.On acinar cells they directly induce cellular and organellar（zymogen granules，mitochondria）injury that leads to intracellular protease activation and cell death.As a consequence cytokines are released and inflammatory cells are attracted aggravating the damage.Besides the effect on acinar cells ethanol and metabolites effect on pancreatic stellate cells（PSCs）causing synthesis of extracellular matrix components（Apte et al.2000；Lugea et al.2003）.
It has attempted to translate the clinical features associated with alcohol consumption into experimental animal models for chronic pancreatitis.Unfortunately the results are very disappointing and so far there is no satisfactory model for chronic pancreatitis using ethanol application alone.Besides the pancreas other organs such as lung and liver are injured by alcohol exposure and animals don't develop proper chronic pancreatitis（Li et al.2008；McIlwrath and Westlund 2015；Schneider et al.2009）.
3.5.1 Duration of Alcohol Application
Technically it is possible to induce chronic pancreatitis in animals with alcohol.Usually alcohol is given together with their daily diet via an oral route often as a Lieber-DeCarli liquid diet.The concentration of ingested ethanol can vary（see below）.In more uncommon experimental setups alcohol is applied by other ways，i.e.via a gastrostomy or directly into the pancreatic duct.
Features of chronic pancreatitis such as acinar cell atrophy and fibrous tissue replacement were achieved by long-term ethanol feeding（up to 30 months）in rats.Pattern of fibrosis resembled those of human chronic pancreatitis（segmental or perilobular distribution）and intraductal protein plugs with partial calcifications were seen as known from humans.Signs of chronic pancreatitis appeared very late when only alcohol is applied and were noticed between the 20th and 30th month of alcohol exposure.By that time already half of the animals died of old age（Sarles et al.1971）.It is quite clear that application of alcohol alone imposes limitations，as long-term treatment and a high death rate are not only time consuming but also questionable from an ethical pathophysiology point of view.
In more recently developed experimental models shorter treatment periods（up to 6 months）were used.However，morphologic changes were less distinct.Secretory enzyme levels behaved differently under chronic ethanol exposure:Amylase secretion was impaired but levels of zymogens such as pro-elastase and（chymo-）trypsinogen were enhanced something normally observed during acute pancreatitis（Li et al.2008；Perkins et al.1995）.When ethanol was injected directly into the pancreatic duct of rats histologic features of chronic disease like interstitial fibrosis，loss of exocrine acinar cells and ductal dilation were observed at quite early time points（7 days）.Limiting factors are the invasive technique and the unphysiologic route of alcohol administration to the animals（Unal et al.2015）.
3.5.2 Techniques of Alcohol Application to Animals
Alcohol supplementation in nutrition needs to exceed a minimum amount so that blood alcohol reaches toxic concentrations in the pancreas.Charles Lieber and Elonore DeCarli contributed much to the development of optimal nutrition formulas for scientific use when they investigated different liquid diets containing ethanol and their effects on organs.The natural aversion of rats against alcohol has been overcome by incorporation of alcohol in a fluid.Usually liquid diets can be prepared more easily than solid foods and are more flexible to adjust depending on the experimental design and the investigator's need.Controls can be generated by replacement of alcohol with other macronutrients such as carbohydrates（Lieber and DeCarli 1989；Lieber et al.1965）.Alcohol concentration in liquids can be calculated according to their proportion of energy supply for total energy intake.In rats an ethanol amount of 5 g/dL or 36%of total energy supply was found to achieve a reasonable blood concentration of at least 20 mM or 100 mg/dL.Lower blood alcohol levels would be ineffective and would not fit to real clinical condition.Higher alcohol concentrations usually will not be consumed by the rat because they dislike its taste（Lieber and DeCarli 1989）.Meanwhile Lieber-DeCarli diets have gained wide acceptance as a standard for scientific use when studying effects of chronic alcohol abuse（Perkins et al.1995）.
Thereare alternative routes such as an application via a gastrostomy or gavage or directly into the ductal system after laparotomy that allows ingestion of high-percentage ethanol diets（Tsukamoto et al.1988）but it remains debatable whether this type of application interferes too much with physiologic intrinsic or extrinsic stimulation of the pancreas.In addition unphysiologic application routes won't mimic human situation and pancreatic injury can arise due to other factors rather than ethanol（Lugea et al.2010）.
Gender of animals affects severity of alcoholinduced injury and therefore should be considered when designing an experiment.Alcohol induced liver injury is greater in females than in males demonstrated by histology and levels of serum transaminases（Iimuro et al.1997）.No direct comparative studies have been performed for evaluation of pancreatic damage so far.When using female rats for experiments pancreatic damage under high-dose ethanol and fat diet occurs earlier at already 8 weeks observation time，indicating that female animals might be more susceptible to pancreatic damage（Kono et al.2001）.
3.5.3 Alcohol Application and Fat
Only a minority of alcoholics（around 5%）ultimately develops chronic pancreatitis so that other co-factors need to be present for manifestation of overt disease.A majority of patients suffered from recurrent acute pancreatitis（RAP）before they come down with chronic pancreatitis.Time to progression varies and lies between 1 and 19 years（median 5.7 years）（Ammann et al.1994）.Alcohol is regarded to be a kind of predisposing or sensitizing agent to the pancreas but other triggers need to be present so that chronic pancreatic will develop（Aghdassi et al.2015；Pandol et al.2011）.(https://www.daowen.com)
One of them is fat.Dietary fat is an important contributor to alcohol-induced pancreatic injury.In the Tsukamoto-French enteral alcohol feeding model rats were fed by a liquid diet containing ethanol and different concentrations of fat.Fat was prepared from corn oil，that is mainly composed of（poly-）unsaturated fatty acids.Diets were classified as low-fat（4-5%of total calory intake），high-fat（22-25%）or extra high-fat（30-35%）diet（Tsukamoto et al.1988）.With increase of dietary fat content there was a potentiation of pancreatic injury:Histopathologic changes included acinar atrophy，a patchy distribution of interstitial fibrosis，fatty replacement and some fat necrosis starting at 4 weeks of diet.Exocrine and endocrine function seemed to be not substantially altered，as plasma trypsinogen and glucose of the“high-fat”dietary group were comparable to controls.Results of high-fat diet were confirmed by further studies and some of them could even show impairment of endocrine function（Mcllwrath and Westlund 2015）.
Otherroutes of combined ethanol and fat administration have been tried.Direct intraperitoenal application of fatty acids and ethanol induced acute pancreatitis in a mouse model.Conversely，co-application of 3-benzyl-6-chloro-2-pyrone（3-BCP），an inhibitor of carboxy ester lipase（CEL），inhibited FAEE production and ameliorated pancreatic damage（Huang et al.2014）.
These results first indicate that a balance of oxidative and non-oxidative metabolization is critical for prevention of pancreatic damage.Preponderance of non-oxidative ethanol metabolism produces FAEEs and both ethanol and fatty acids exert toxic effects on the pancreas.An exogenous administration of fatty acids increases FAEE amounts and thus pancreatic damage.Secondly carboxy ester lipase（CEL）was identified to be one of the enzymes responsible for the damage.Notably，long-term ethanol feeding even increases pancreatic CEL activity（Pfutzer et al.2002；Criddle 2015）.On a cellular level FAEEsinduced sustained high Ca2+ elevations that lead to uncontrolled trypsinogen activation（Gerasimenko et al.2009）.Moreover there is a loss of mitochondrial function by opening the mitochondrial transition pore leading to a fall of intracellular ATP levels that is a prerequisite for the emergence of necrosis（Criddle et al.2006）.
Pancreatic injury that is caused by high doses of ethanol and a fatty diet is less severe when medium-chain triglycerides，i.e.saturated fatty acids，are used instead of unsaturated fats（Kono et al.2001）.Less steatosis of the pancreas as well as reduced inflammatory infiltration and necrosis were observed.One of the underlying mechanisms might be a reduction of lipid peroxidation and consecutively a prevention of free radical formation in presence of MCT-fats.
3.5.4 Ethanol，Cholecystokinin and Chronic Pancreatitis
From ex-vivo experiments with isolated acinar cells and animal models it is well known that acute pancreatitis can be induced by repetitive applications of supraphysiologic doses of cholinergic and cholecystokinin（CCK）agonists in rodents（Lugea et al.2010；Halangk et al.2000）.Therefore combinations of alcohol feeding with caerulein injections either as a single shot or by repetitive applications were thought to be an attractive way for induction of chronic pancreatitis（Lugea et al.2010；Deng et al.2005；Gukovsky et al.2008；Perides et al.2005）.Usually ethanol feeding is performed prior to caerulein applications and lasts for approximately 2-8 weeks.Results consistently showed histopathologic changes compatible with chronic pancreatitis such as activation of pancreatic stellate cells with an increase of collagen content and fibrosis.Combination of alcohol and caerulein clearly enhanced fibrosis formation whereas alcohol alone only led to an inflammatory reaction（Deng et al.2005；Perides et al.2005）.Exocrine insufficiency was not observed.
Usually a concentration of 50 µg/kg body weight is used for induction of acute pancreatitis but this dose can be markedly reduced（up to 0.5 µg/kg）when animals have been fed with an ethanol-diet before.This drastic reduction again underlines the ethanol sensitizing effect for other harmful stimuli.
3.5.5 Combination of Ethanol and Other Agents
Further agents have been used in combination with ethanol with promising results.Lipopolysaccharides（LPS）are known as endotoxins，cause activation of pancreatic stellate cells and inhibit apoptosis of PSCs so that extracellular matrix proteins will be increasingly synthesized and released.Mice fed with ethanol and subjected to（intraperitoneal）LPS injections showed morphological signs of chronic pancreatitis whereas ethanol alone was ineffective（Nakayama et al.2014）.Changes were detectable after 6 weeks of LPS treatment.Intraductal bile salt infusions to rats that received both a Lieber DeCarli diet and caerulein injections developed severe necrotizing pancreatitis with concomitant lung injury（Schneider et al.2009）.
Cyclosporin A is an immunosuppressant that is clinically used for suppression of organ transplant rejection.Simultaneous treatment of rats with caerulein and daily cyclosporin led to acute pancreatitis without any regeneration of the organ.This distortion of the repair mechanism caused myofibroblast proliferation，collagen production and fibrosis as seen in chronic pancreatitis（Vaquero et al.1999）.In a modified experimental model when rats were pre-fed with an ethanol containing Lieber DeCarli diet（36%of total calorie intake）pancreatic damage was aggravated with enhanced fibrosis formation，acinar tissue loss and sustained inflammatory infiltration（Gukovsky et al.2008）.It is still unclear whether these models will be useful for investigation of exocrine or endocrine deficiencies，as well.
In other experimental settings alcohol feeding was performed after preceding interventions.Trinitrobenzene sulfonic acid（TNBS）is a chemical hapten that binds to tissue proteins capable of inducing T-cell mediated immunity and generation of oxygen radicals and other inflammatory mediators（Tatsumi and Lichtenberger 1996）.This compound has already been used in rats for experimental models of colitis and cholangitis.A retrograde instillation of TNBS into the pancreatic ductal system causes fibrotic change of the organ and continuous weight loss.Endocrine insufficiency was not observed（Puig-Divi et al.1996）.When rats were fed with ethanol for another 2-4 weeks fibrosis and glandular atrophy were more pronounced and animals now showed an impaired glucose tolerance indicating beginning of endocrine insufficiency（Puig-Divi et al.1999）.