Role of bone marrow cells in the development of pancreatic fibrosis in a rat model of pancreatitis induced by a choline-deficient/ethionine-supplemented diet.

Bone marrow cell (BMC)-derived myofibroblast-like cells have been reported in various organs, including the pancreas. However, the contribution of these cells to pancreatic fibrosis has not been fully discussed. The present study examined the possible involvement of pancreatic stellate cells (PSCs) originating from BMCs in the development of pancreatic fibrosis in a clinically relevant rat model of acute pancreatitis induced by a choline-deficient/ethionine-supplemented (CDE) diet. BMCs from female transgenic mice ubiquitously expressing green fluorescent protein (GFP) were transplanted into lethally irradiated male rats. Once chimerism was established, acute pancreatitis was induced by a CDE diet. Chronological changes in the number of PSCs originating from the donor BMCs were examined using double immunofluorescence for GFP and markers for PSCs, such as desmin and alpha smooth muscle actin (αSMA), 1, 3 and 8 weeks after the initiation of CDE feeding. We also used immunohistochemical staining to evaluate whether the PSCs from the BMCs produce growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF) ß1. The percentage of BMC-derived activated PSCs increased significantly, peaking after 1 week of CDE treatment (accounting for 23.3±0.9% of the total population of activated PSCs) and then decreasing. These cells produced both PDGF and TGFß1 during the early stage of pancreatic fibrosis. Our results suggest that PSCs originating from BMCs contribute mainly to the early stage of pancreatic injury, at least in part, by producing growth factors in a rat CDE diet-induced pancreatitis model.

Chronic pancreatitis in mice by treatment with choline-deficient ethionine-supplemented diet.

Although chronic pancreatitis is a risk factor for pancreatic ductal adenocarcinoma (PDA), the relationship between chronic pancreatitis and PDA remains obscure. A critical obstacle to understanding the role of chronic pancreatitis is the lack of animal models. To develop one such model, mice were fed long-term with a choline deficient ethionine-supplemented (CDE) diet. Histological evaluation revealed that chronic pancreatitis, characterized by acinar atrophy, fibrosis and well-developed tubular complexes (TCs), was observed after 24 weeks of CDE diet treatment. Furthermore, expression of epidermal growth factor receptor (EGFR) and its ligands; serine protease inhibitor Kazal type 3 (Spink3) and transforming growth factor alpha (TGF alpha) and activation of K-Ras (GTP-Ras formation), which are frequently observed in human PDA, were indeed observed in parallel with TCs formation. Neoplastic lesions were not found after 54 weeks of treatment, suggesting that a continuation of CDE diet or another insult is required for the development of PDA.

Expression of extracellular matrix genes in cultured hepatic oval cells: an origin of hepatic stellate cells through transforming growth factor beta?

BACKGROUND: Hepatic oval cells, progenitor cells in the liver, can differentiate into hepatocytes and bile duct cells both in vitro and in vivo. Although hepatic stellate cells are another important cell component in the liver, less attention has been focused on the relationship between hepatic oval cells and hepatic stellate cells. METHODS: Hepatic oval cells were isolated from rats fed a choline-deficient diet supplemented with 0.1% ethionine for 6 weeks and characterized by electron microscopy, flow cytometry, reverse transcription polymerase chain reaction, Western blot and bi-direction differentiation. After treatment with transforming growth factor-beta1 (TGF-beta1), changes in cell viability, morphology, extracellular matrix (ECM) expression and immune phenotype were analysed in these cultured and adherent hepatic oval cells. RESULTS: The primary cultured hepatic oval cells were positive for the oval cell-specific markers OV-6, BD-1/BD-2 and M2PK as well as the hepatocyte markers albumin and alpha-foetoprotein. These hepatic oval cells differentiated bipotentially into hepatocytes or bile duct-like cells under appropriate conditions. It is noteworthy that these bipotential hepatic oval cells expressed ECM genes stably, including collagens, matrix metalloproteinases and tissue inhibitor of mellatoproteinase. Furthermore, except for growth inhibition and morphological changes in the hepatic oval cells after exposure to TGF-beta1, there was an increased expression of ECM genes, the onset expression of snail and loss expression of E-cadherin. During this process, TGF-beta1 treatment induced an upregulation of marker genes for hepatic stellate cells in hepatic oval cells, such as desmin and GFAP. CONCLUSION: Except for the expression of ECM, the cultured hepatic oval cells could induce an increased expression of hepatic stellate cell markers by TGF-beta1 through an epithelial-mesenchymal transition process, which might indicate the contribution of hepatic oval cells to liver fibrosis.

Risperidone attenuates local and systemic inflammatory responses to ameliorate diet-induced severe necrotic pancreatitis in mice: it may provide a new therapy for acute pancreatitis.

In a previous article, we showed that a potent serotonin-, 5-hydroxytryptamine-2A (5-HT(2A)) antagonist, risperidone, ameliorated cerulein-induced edematous pancreatitis in mice. In the present article, young female mice were fed a choline-deficient, ethionine-supplemented diet. All of the mice developed severe necrotic pancreatitis, and approximately 50% of them died within 4 days. Serum levels of proinflammatory interleukin (IL)-6 significantly increased on day 3 and returned toward the control on day 4 of choline-deficient ethionine-supplemented (CDE) diet treatment. The time course of IL-6 levels paralleled those of plasma amylase and lipase activities. On the other hand, platelet counts significantly decreased on day 3, and the change became more marked on day 4, coinciding with mortality and histological alterations of the pancreas (edema, inflammatory cell infiltration, necrosis). Preceding these changes, plasma levels of 5-hydroxyindoleacetic acid (5-HIAA) increased on feeding a CDE diet to reach a peak on day 3 and returned toward the control on day 4. Risperidone (0.1-3.2 mg/kg twice a day) hardly affected the 5-HIAA levels but dose-dependently attenuated the serum IL-6 levels, plasma amylase/lipase levels, platelet counts, histological alterations, and mortality of diet-induced pancreatitis mice. These results are discussed in relation to the pathogenesis of acute pancreatitis. Thus, we speculate that acinar cell injury triggers local inflammatory reactions and, if coincided with enhanced IL-6 release, leads to a systemic inflammatory response syndrome, which is responsible for the mortality. In addition, it is suggested that diet-induced 5-HT release and 5-HT(2A) receptor activation are involved in this whole process of pancreatitis development. Risperidone may provide a new therapy for the disease.

Bone marrow cells play only a very minor role in chronic liver regeneration induced by a choline-deficient, ethionine-supplemented diet.

Liver progenitor (oval) cells have enormous potential in the treatment of patients with liver disease using a cell therapy approach, but their use is limited by their scarcity and the number of donor livers from which they can be derived. Bone marrow may be a suitable source. Previously the derivation of oval cells from bone marrow was examined in rodents using hepatotoxins and partial hepatectomy to create liver damage. These protocols induce oval cell proliferation; however, they do not produce the disease conditions that occur in humans. In this study we have used the choline-deficient, ethionine-supplemented (CDE) diet (which causes fatty liver) and viral hepatitis as models of chronic injury to evaluate the contribution of bone marrow cells to oval cells under conditions that closely mimic human liver disease pathophysiology. Following transplantation of lacZ-transgenic bone marrow cells into congenic mice, liver injury was induced and the movement of bone marrow cells to the liver monitored. Bone marrow-derived oval cells were observed in response to the CDE diet and viral injury but represented a minor fraction (0-1.6%) of the oval cell compartment, regardless of injury severity. In all situations only rare, individual bone marrow-derived oval cells were observed. We hypothesized that the bone marrow cells may replenish oval cells that are expended by protracted liver injury and regeneration; however, experiments involving a subsequent episode of chronic liver injury failed to induce proliferation of the bone marrow-derived oval cells that appeared as a result of the first episode. Bone marrow-derived hepatocytes were also observed in all injury models and controls at a frequency unrelated to that of oval cells. We conclude that during viral-and steatosis-induced liver disease the contribution of bone marrow cells to hepatocytes, either via oval cells or by independent mechanisms, is minimal and that the majority of oval cells responding to this injury are sourced from the liver.

Pancreatic concentrations of cefepime in experimental necrotizing pancreatitis.

To evaluate the penetration of cefepime in the inflamed pancreas, three doses of 50 mg/kg were administered intramuscularly at 8-h intervals after induction of acute necrotizing pancreatitis using intraperitoneal injection of DL-ethionine in 35 rabbits and in 33 controls. Animals were sacrificed and concentrations of cefepime were determined by a microbiological assay. Cefepime reached its peak concentrations 60 min after the last drug dose when mean values of 46.05 microg/ml, 22.34 microg/g and 34.74 microg/ml were found in serum, pancreas and bile, respectively, in rabbits with acute necrotizing pancreatitis and 45.19 microg/ml, 12.68 microg/g and 20.77 microg/ml respectively in controls. Tissue/serum ratios of cefepime were 0.48, 0.23, 0.15 and 0.09 at 60, 90, 120 and 180 min, respectively, after the last dose of cefepime in rabbits with acute necrotizing pancreatitis and 0.28, 0.18, 0.16 and 0.16, respectively at 60, 90, 120 and 180 min in controls. It is concluded that the administration of cefepime in rabbits with acute necrotizing pancreatitis resulted in pancreatic tissue levels well above the MIC90s of the common pathogens involved in pancreatic superinfection, so that its administration might be proposed for the therapy of superinfection following acute necrotizing pancreatitis in humans.

Effect of d,l-ethionine administration on the histomorphology of canine pancreatic acinar and beta-cells.

d,l-Ethionine produces pancreatic exocrine necrosis and islet proliferation in hamsters and dogs. As a first step in examining whether induction of islet proliferation has therapeutic applications in animals with exhausted or destroyed insulin-producing beta-cells, we studied pancreatic pathological alterations after intravenous administration of d,l-ethionine in normal dogs. Histomorphological changes in pancreatic acinar cells and beta-cells were assessed in three groups of six clinically normal crossbred dogs administered d,l-ethionine (100 mg/ kg) intravenously three times a week for two weeks. Six additional dogs served as untreated controls. Group I was euthanased and necropsied on day 15 (72 hours after the final dose of ethionine). Groups II and III were euthanased on days 29 and 43 respectively. Severe acinar destruction occurred resulting in significant (p < 0.001) shrinkage of the pancreas in all groups. Although there was variability in histomorphology within groups, pancreases of group I generally exhibited widespread loss of pancreatic acinar structure. Remaining acinar cells were difficult to discern from other cell types within lobules and were surrounded by infiltrates, predominantly of lymphocytes. Partial acinar cellular regeneration had occurred by day 29, but was still incomplete at day 43. Immunohistochemistry suggested that the effect of d,l-ethionine administration on the histomorphology of beta-cells in the left lobe was minimal; however, morphometry demonstrated a significant (p < 0.05) increase in the number of individual beta-cells in groups II and III, and clusters of 2-10, 10-20 and 20+ cells in Group II. It is probable that the apparent increase in the number of individual and other beta-cell arrangements observed in some groups resulted primarily from alterations in the exocrine tissue, although some beta-cell hyperplasia cannot be excluded completely.

Evaluation of d,l-ethionine as a mechanism for pancreatic islet regeneration in dogs.

OBJECTIVE: To determine whether induction of pancreatic necrosis and islet proliferation by d,l-ethionine has potential for treating dogs with beta-cell insufficiency. DESIGN: Eighteen mixed breed dogs of both sexes were given d,l-ethionine at 100 mg/kg three times weekly for 2 weeks; 6 dogs were euthanased at 2, 14 and 28 d after the last dose. METHODS: Clinical signs during administration and recovery were assessed. Routine biochemical analyses were performed before each ethionine dose and then once weekly. Faecal samples were examined weekly for malassimilated nutrients and blood. Blood coagulation screening tests (OSPT and APTT) were determined on four dogs after ethionine administration. Intravenous glucose tolerance tests were conducted before the first and after the last ethionine dose and then fortnightly. All dogs were necropsied and pancreas, liver, kidney and jejunum were examined microscopically. RESULTS: During ethionine administration all animals displayed vomiting, inappetence, diarrhoea (often with blood), weight loss and depression. Three dogs were euthanased prematurely due to severe illness, but those allowed to recover were eating and brighter 7 d after cessation of ethionine administration. Serum concentrations of TLI, amylase and lipase increased initially, then decreased, during administration but retumed to normal during recovery. Concentrations of ALT, ALP, unconjugated and conjugated bilirubin increased during administration then decreased slowly. Histological examination revealed hepatic lipidosis and necrosis, but no renal or jejunal lesions. In most dogs, faecal examination demonstrated increased undigested starch and muscle, as well as increased digested and undigested fat, during ethionine administration or early during the recovery period, suggesting transient malassimilation. APTT was unchanged but OSPT was prolonged in all dogs. There was no impairment of insulin secretion or glucose intolerance and C-peptide concentrations were unaffected. Immediately after ethionine administration there was delayed insulin degradation and by day 43 there was evidence of increased insulin sensitivity. CONCLUSION: d,l-ethionine administration in dogs appeared not to interfere with insulin secretion, but caused clinical signs and laboratory changes indicative of pancreatic exocrine necrosis, severe hepatobiliary disease and transient malassimilation. Pancreatic and hepatic dysfunction was severe but clinical recovery occurred after ethionine administration ceased. The severe side-effects observed with d,l-ethionine should preclude its potential use for treating diabetes mellitus in dogs.

CT with the hepatocyte-specific contrast medium FP 736-04 in an experimental model of liver steatosis.

PURPOSE: To investigate whether liver uptake of the iodinated hepatocyte-specific lipid emulsion FP 736-04 is altered by fatty infiltration of the liver. MATERIAL AND METHODS: Fatty infiltration of the liver was induced in female Sprague-Dawley rats by an intraperitoneal injection of L-ethionine preceded by 15 h of food withdrawal. CT of the liver was performed before and 24 h after the administration of L-ethionine and, in addition at the latter time point, after an i.v. injection of 1.0 ml/kg b.w. of FP 736-04. A control group was subjected to the same CT examination protocol. RESULTS: Intraperitoneal administration of L-ethionine caused liver steatosis, as established by liver triglyceride analysis, leading to a significant decrease in the liver attenuation, from 69.2 +/- 2.4 to 27.8 +/- 12.0 HU. The uptake of FP 736-04 by the fatty liver was significantly reduced, yielding a maximum enhancement of 22.3 +/- 7.9 HU as compared to a value of 32.3 +/- 5.0 HU in the control group. CONCLUSION: Enhancement by FP 736-04 was reduced in the steatotic liver compared with the normal liver. It remains to be established whether this degree of enhancement is sufficient for reliable lesion detection.

How and where does acute pancreatitis begin?

Circumstantial evidence suggests that gallstone-induced pancreatitis is triggered by obstruction of the pancreatic duct. In this report I will review the results of studies conducted during the last decade that have employed the diet-induced, secretagogue-induced, and duct obstruction-induced models of experimental pancreatitis to investigate the early events that lead to the development of acute pancreatitis. In each of these models, digestive enzyme zymogens and the lysosomal hydrolase cathepsin B were found to become colocalized. These observations have led to the hypothesis that intra-acinar cell activation of digestive enzyme zymogens by lysosomal hydrolases may be an important critical event in the development of acute pancreatitis. Recent morphologic studies evaluating the initial 24 hours after ligation of the opossum pancreatic duct indicate that the earliest lesions in this model of hemorrhagic pancreatitis occur in acinar cells.

[Study on the trophic effect of camostate mesilate on ethionine-induced pancreatic injury rat].

Pancreatic injury was induced to rats with intraperitoneal injection of ethionine 60 mg per 100 g BW twice or three times weekly for 6 weeks. These rats were given 100 mg/kg of Camostate mesilate (CM) via a gastric tube daily for 14 days. CM administration resulted in an increase of pancreatic wet weight, hypertrophy and hyperplasia of acinar cells, and an increase of exocrine pancreatic function. Acini prepared from CM and ethionine-treated rats exhibited increased response to caerulein, but decreased sensitivity to caerulein. The plasma CCK level in rats with CM administration 24 hours later was higher than that without CM administration. However, there were no significant changes in plasma CCK and secretin level thereafter. We concluded that CM had a trophic effect on the pancrease with ethionine-induced pancreatic injury, and CCK was considered playing the same role in injured pancreas as the normal rat pancreas. Studies using CCK receptor antagonist are needed for further clarification.

Efecto de la etionina sobre el hígado de rata en condiciones de diferente aporte de proteina en la dieta / Ethionine effects on rat liver under different protein dietary contents

Durante el desarrollo de la vida de un ser vivo reviste sinbgular importancia la capacidad de adaptación de su organismo a los cambios cambientales. Esta adaptabilidad parece depender en grado sumo del aporte de proteínas en la alimentación. Por ejemplo, la deficiencia proteínica en la dieta provoca un marcado descenso de las defensas del organismo, elevando a la vez su susceptibilidad a los agentes tóxicos ambientales cuyo número y variedad actualmente cada vez es mayor. En estas condiciones es clara la necesidad de la fórmula óptima alimentaria como media de protección fisiológico frente a un agresivo entorno. Este trabajo es un grano de arenas, camino de esta búsqueda, el cual se realizó en animales de experimentación (ratas) a nivel subcelular (lisosoma), abarcando dos aspectos: morfológico y enzimático (enzimas marcadoras-fosfatasa ácida, ribonucleasa ácida y catepsina). Se encontró: la etionina en condiciones de alimentación con aporte normal proteínico (18.5%) provoca alteraciones leves en los hepatocitos (infiltración grasa periportal, mínimas alteraciones del patrón enzimático y de la permeabilidad celular). En condiciones de alimentación deficiente en proteínas la manifestación histológica es más severas (disminución de ARN celular, infiltración grasa difusa), siendo mínimo el cambio en el patrón enzimático y en la permeabilidad celular. En condiciones de alimentación con exceso de proteína (44.5%) los cambios histológicos y de permeabilidad son discretos y se registró una tendencia a la transformación favorable anabólica patrón enzimático. Se concluye: el aporte elevado de proteína en la dieta surte en ratas el efecto de antídoto de la acción hepatotóxica de la etionina

Cirrhosis of the liver: a reversible disease?

This paper is a speculative review of the irreversibility of cirrhosis of the liver. Experimental studies dealing with the specific issue of irreversibility of the process are summarized, and the following three general propositions are derived: 1. All experimental models of cirrhosis are reversible, provided the inciting agent is removed and sufficient time is allowed for the return to normal liver structure. 2. Experimental cirrhosis of the liver goes through two successive stages, differing (among many other features) in the time and completeness of their reversibility. 3. Increased reticulum fibers are more easily and completely resorbed than thick collagenous bundles. Human cases of cirrhosis of the liver in which the fact of regression appears to be sufficiently documented are also summarized. Most of them seem to fulfil the three conditions derived from the analysis of experimental data, namely, withdrawal of the etiologic agent at an early stage in their development (with a predominance of reticulum over collagen fibers) and allowance of sufficient time to document the disappearance of the disease. Experimental studies on the mechanisms of collagen degradation in general, and in the liver of mammals in particular, are also reviewed. It is concluded that much remains to be done but that the outcome is by no means hopeless.

Increased alpha-fetoprotein biosynthesis in rats following DL-ethionine and carbon tetrachloride injuries.

The incorporation of 14C-leucine into alpha-fetoprotein (AFP) in serum and liver extracts from DL-ethionine and carbon tetrachloride (CCl4)-injured rats was measured directly by using an immunochemical technique. The maximum value with nearly 10-fold increases of serum AFP was obtained in 3 days of both treatments. However, no significant increase in the immunoprecipitable AFP in liver extracts was detected under these conditions.

Influence of dietary factors on the pancreatotoxicity of ethionine.

Choline deficiency enhances greatly the pancreatotoxicity of DL-ethionine. Mice fed DL-ethionine with a choline-deficient diet develop a fatal acute hemorrhagic pancreatic necrosis with fat necrosis (AHPN) in 5 days. Induction of the AHPN is completely prevented by dietary methionine and drastically reduced by dietary methionine and drastically reduced by dietary choline. The amount of proteins, carbohydrates, and fat in the diet influences its consumption by the animals and thus the severity of the pancreatic pathology. The histogenesis of the process is characterized by widespread alterations of the membranous organelles of the acinar cells, especially the endoplasmic reticulum and the zymogen granules. The onset of the hemorrhagic necrosis of the pancreas is due to an endogenous intraparenchymal activation of the zymogenic proteases, including proelastase. The new experimental model of AHPN appears to mimic very closely the clinical course, the anatomic pathologic lesions, and the postulated pathogenesis of the corresponding human disease.

Acute hemorrhagic pancreatic necrosis in mice. Influence of the age and sex of the animals and of dietary ethionine, choline, methionine, and adenine sulfate.

The influence of several factors on the development of acute hemorrhagic pancreatitis (pancreatic necrosis) with fat necrosis in mice fed DL-ethionine with a choline-deficient diet has been investigated. The results showed that: a) the incidence of the induced disease is dependent upon the age and sex of the animals and the dietary level of ethionine; b) 100% of young female mice fed 0.5% ethionine develop acute hemorrhagic pancreatitis and die within 5 days; c) the incidence is only 10 to 20% when 0.5% ethionine is fed with either a choline-supplemented diet or with laboratory chow; and d) development of pancreatic pathology is completely prevented by the inclusion in the diet of 0.5% methionine but not by the inclusion of 0.5% adenine. Possible mechanisms whereby choline deficiency potentiates the pancreatotoxicity of ethionine in mice are discussed.