Animal Models of Pancreatitis

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As mentioned above, the mechanisms responsible for pancreatitis in humans are complex. Animal models can reflect only part of them. However, they may provide useful information, especially when the models are carefully selected in accordance with the experimental purpose or design. Therefore, it is necessary that researchers have a sufficient knowledge of the characteristics of the various models.

In the subsections below we describe the pancreatic histology observed in our experiments using various animal models. First, we focus on the characteristics of pancreatic fibrosis. Later, the results of animal models which unfortunately failed to form fibrosis under our experimental conditions are also mentioned, since the models have been most often used in the studies on pancreatitis.

Pancreatitis in Male WBN/Kob Rats

Male WBN/Kob rats show hemorrhage and edema in intralobular and interlobular areas at the age of about 2 or 3 months, as well as widespread acute pancreatitis with slight fibrosis (fig. 1a). At 4 months of age, the fibrosis becomes marked (fig. 1b). Because of these features, this rat strain is often used as a chronic pancreatitis model [62-65]. In some reports on acute pancreatitis in this strain, initial bleeding was thought to arise around the islets of Langerhans [65]; however, we could not identify the site of bleeding in our studies [66]. Female WBN/Kob rats show no changes in their pancreas; therefore, sex hormones

Fig. 1. Characteristics of spontaneously occurring pancreatitis in WBN/Kob rats. a Pancreas from male WBN/Kob rat at 2 months of age. Interlobular hemorrhage and edema are seen. b Pancreas from male WBN/Kob rat at 4 months of age. Inter- and intralobular fibrotic changes are seen. Destruction of pancreatic islets has occurred as the result of the widespread fibrosis. c Pancreas from male WBN/Kob rat at 8 months of age. Inflammatory changes have continued, while the fibrosis shows a tendency toward resolution.

Fig. 1. Characteristics of spontaneously occurring pancreatitis in WBN/Kob rats. a Pancreas from male WBN/Kob rat at 2 months of age. Interlobular hemorrhage and edema are seen. b Pancreas from male WBN/Kob rat at 4 months of age. Inter- and intralobular fibrotic changes are seen. Destruction of pancreatic islets has occurred as the result of the widespread fibrosis. c Pancreas from male WBN/Kob rat at 8 months of age. Inflammatory changes have continued, while the fibrosis shows a tendency toward resolution.

must be involved in the occurrence of pancreatitis. In the males, fibrosis was prominent in the periductal or interlobular areas, extended into the intralobular area, and disrupted the islet tissues. This fibrotic change is thought to be why diabetes occurs in male WBN/Kob rats. Unfortunately, fibrosis in this strain is also reversible; in other words, the fibrosis reaches a peak at 4 months of age and gradually decreases thereafter (fig. 1c). This model can thus not be considered a suitable model for human chronic pancreatitis. In contrast, we investigated WBN/Kob rats, taking their fibrous characteristics into account, and found some factors related to the formation and resolution of the fibrosis (described later).

In addition to other minor changes, we also observed signs of apoptosis [67, 68] in acinar cells and inflammatory cells in the fibrotic and inflamed area. Protein plugs were not observed during the acute phase (about 2 months of age) but were recognized in inflamed areas from 4 months of age onwards.

Caerulein-Induced Pancreatitis

Caerulein, which structurally resembles cholecystokinin (CCK) and gastrin, is known to exert pharmacological effects similar to those of CCK [69-73]. Therefore, this model has often been used to clarify the pathogenetic mechanism of pancreatitis in relation to metabolism [72, 74, 75]. Various administration routes and doses have been selected in many investigations and on the whole the results indicate that caerulein can induce acute pancreatitis.

In our experiments, caerulein-treated mice were used to investigate the relationship between the histopathologic severity of pancreatitis, presence or absence of calculi, and dosage frequency. Ten-week old C57BL6 mice were intraperitoneally injected with 100 |xg/5 ml/kg of caerulein 4, 7, 10 or 13 times at 1-hour intervals, and sacrificed at 7, 10, 13, or 24 h or 2, 3 or 8 days after the last injection. In the mice dosed 4 times, inflammatory changes were comparatively slight and there were almost no advanced changes in the pancreas (fig. 2b). On the other hand, the histopathology of mice treated with 7 doses showed massive and severe edema, necrosis of acinar cells, and hemorrhage, and resembled that of mice treated with 10 (fig. 2c) or 13 doses (fig. 2d), when the animals in these groups were examined 13 h after the final dosing. However, at 8 days after the final dosing, there were great differences in the changes between the mice treated with 7 or 10 doses and those dosed 13 times. At that point, the mice treated 7 or 10 (fig. 2e) times showed slight changes, i.e. slight necrosis of acinar cells with low-grade inflammation and ductal proliferation. In contrast, mice treated 13 times showed more severe necrosis of acinar cells, inflammatory cell infiltration, ductal hyperplasia, and fibrosis (fig. 2f). Interestingly, many calculi were observed in the pancreatic ducts from the mice treated with 13 doses. It is more than probable that calculi formation, which was not clear at 13 h after the final dosing, occurred later, and induced severe pancreatic changes in the animals treated with 13 doses. This hypothesis may indicate that the cause of caerulein-induced pancreatitis is related to acceleration of pancreatic secretion and autolysis. Furthermore, the formation of calculi may be a significant step toward the development of pathological features including fibrosis [76-78].

Pancreatitis in the DBTC Model

In recent years, there have appeared many reports on models of pancreatitis induced by dibutyltin dichloride (DBTC) [79-82]. Rats or mice treated

Fig. 2. Characteristics of cerulein-induced pancreatitis. a Pancreas from control mice. No abnormalities are seen. b Pancreas at 13 h after 4 treatments with 100 ^g/kg. Slight infiltration by inflammatory cells is noted. Edema and necrosis are seen. c Pancreas at 13h after 10 treatments with 100 ^g/kg. Severe edema has occurred. Necrosis and inflammatory cell infiltration are also observed. d Pancreas at 13 h after 13 treatments with 100 ^g/kg. Edema, inflammation, and necrosis are at the same levels as in (c). e Pancreas at 7 days after 10 treatments with

Fig. 2. Characteristics of cerulein-induced pancreatitis. a Pancreas from control mice. No abnormalities are seen. b Pancreas at 13 h after 4 treatments with 100 ^g/kg. Slight infiltration by inflammatory cells is noted. Edema and necrosis are seen. c Pancreas at 13h after 10 treatments with 100 ^g/kg. Severe edema has occurred. Necrosis and inflammatory cell infiltration are also observed. d Pancreas at 13 h after 13 treatments with 100 ^g/kg. Edema, inflammation, and necrosis are at the same levels as in (c). e Pancreas at 7 days after 10 treatments with intravenously with DBTC were reported to show epithelial lesions in the extra-hepatic bile duct, leading to chronic inflammation and cystic dilation of the bile duct. Also, tri- and dibutyltin or diethyltin diiodide compounds, which are alkylstannane compounds like DBTC, induce similar lesions in rats or mice, but not in cats, guinea pigs or rabbits [83, 84]. These facts may suggest that the involved cause for the lesion may be partly due to the anatomical character of the pancreaticobiliary duct, since the pancreatic duct and bile duct normally become confluent in rats and mice [85].

In our study, Lewis rats were injected intravenously at a single dose of 8mg/kg of DBTC, and histopathologically examined. Also in our case, cystic dilation of the bile duct was marked several days after treatment. Necrosis and desquamation of bile duct epithelial cells, severe edema and inflammatory cell infiltration around the bile duct, and obstruction of the pancreaticobiliary duct by debris preceded the cystic changes. In the pancreas from mice with a cystic bile duct, scattered colliquative necrosis of the ductal epithelium and peripheral tissue, as well as fatty necrosis, was observed at 1 day after treatment (fig. 3a). Unlike those in ethionine- or arginine-induced pancreatitis (accumulation of secretion components in acinar cells or self-digestion, as described later), the lesions in the DBTC model were localized around the pancreatic ducts, interlobular spaces, and also inconspicuously periductule sites in the intralobular areas (fig. 3b). It was thus probable that the starting point for pancreatitis in the DBTC model was leakage of pancreatic juice or bile into the periductal space, which occurs as a sequela of the destruction of the ductal wall. In the chronic situation, acinar cells became atrophied, and ductal proliferation and fibrosis appeared (fig. 3c, d). In the liver (fig. 3e), it was not surprising that proliferation of bile ducts occurred.

Interestingly, fibrosis in the DBTC model persists for a comparatively long time around the pancreatic ducts and interlobular spaces (fig. 3f). It is just conceivable that the persistent fibrosis may be attributed to fibers composed chiefly of type I collagen, which is known to be much more stable than type III collagen (described later). For instance, fibrosis in male WBN/Kob rats is due to type III collagen, and is known to disappear spontaneously.

100 ^g/kg. Inflammatory changes are mild or nonexistent. Evidence of sporadic regenerated acinar cells is present. f Pancreas at 7 days after 13 treatments with 100 ^g/kg. Unlike in the case of 10 treatments (e), inflammatory changes have progressed. Decrease in acinar cells with severe proliferation of pancreatic ducts is noteworthy. Protein plugs are seen in the pancreatic ducts, which may indicate that such plugs or calculi are factors that exacerbate or reactivate the pancreatitis. Areas of fibrosis have formed in the spaces between the proliferative ducts.

Fig. 3. Characteristics of DBTC-induced pancreatitis. a Pancreas at 1 day after treatment with 8mg/kg. Necrotic epithelium of the pancreatic duct is seen. Acinar cells and adipose tissue around the duct have also become necrotic. Hemorrhage and inflammatory cells are observed. b Pancreas from the same animal as (a). Focal necrosis is also seen in the intralobular areas.These areas are probably located around the intralobular pancreatic ducts. c Pancreas

Fig. 3. Characteristics of DBTC-induced pancreatitis. a Pancreas at 1 day after treatment with 8mg/kg. Necrotic epithelium of the pancreatic duct is seen. Acinar cells and adipose tissue around the duct have also become necrotic. Hemorrhage and inflammatory cells are observed. b Pancreas from the same animal as (a). Focal necrosis is also seen in the intralobular areas.These areas are probably located around the intralobular pancreatic ducts. c Pancreas

It must be noted that in this model there are individual differences between rats. In our experiments, only approximately half of the treated rats developed pancreatitis. So, especially in therapeutic experiments, preconsideration of progress levels of pancreatic lesions in individual animals is necessary before any procedures are started. Finally, we also believe this to be suitable model for congenital cystic dilatation of the common bile duct in humans.

Pancreatitis in the Duct-ligation Model

Pancreatic ducts in the rat are composed of several large ducts and numerous narrow ones, and they open directly into the common bile duct without forming main or accessory pancreatic ducts. Also, the rat pancreas is segmented by the pathways of its ducts and blood vessels into splenic, duodenal, gastric, and parabiliary segments [85]. Various methods for ligation of the pancreatic duct were reported years ago [86-91]. For study purposes, complete or partial ligation was performed at various segments of the pancreas, and duct obstruction was concluded to be the cause of pancreatitis [88, 91, 92].

As described, the pancreatic duct and bile duct of the rat are interfluent outside the duodenum. So, it was thus expected that ligation closer to the duodenum would induce influx of bile into the pancreatic duct. In a study using the rat ligation model, we investigated the relationship between human anomalous arrangement of the pancreaticobiliary duct and pancreatitis; therefore, ligation was done at a position closer to the duodenum.

As a result, dilation of the pancreatic ducts was remarkable in both inter-and intralobular areas. Severe inflammation was observed in the intralobular and periductal spaces. Fatty necrosis was also scattered (fig. 4a, b). At 7 days after ligation, atrophy of acinar cells, proliferation of intralobular pancreatic ducts, and periductal fibrosis were seen (fig. 4c).

Moreover, the cystic dilation of the bile duct was remarkable . It was clear that pancreatic juice flowed into the bile duct, since the contents of the cyst showed amylase activity. On the other hand, bile was not morphologically detected in pancreas specimens in our experiments, and hence there was no evidence of influx of bile into the pancreas. The main causes of occurrence and progression of pancreatitis in the ligation model, as well as in the DBTC model, may therefore be elevation of ductal pressure, and not bile influx into the pancreatic duct.

at 3 days after treatment. Necrosis, infiltration by fibroblastic cells and proliferation of pancreatic ducts have progressed. d Pancreas at 2 weeks after treatment. Fibrosis has become firm by this time. Regenerative epithelial cells are arranged at the luminal surface of the pancreatic ducts. e Liver at week 2 after treatment. Evidence of proliferation of the bile duct with slight infiltration of inflammatory cells is seen. f Pancreas at 24 weeks after treatment. Extensive fibrosis is seen around the ducts. Fibrosis has expanded into the intralobular spaces.

Fig. 4. Characteristics of pancreaticobiliary duct-ligation model. a Pancreas at 3 days after ligation near the duodenum. Drastic changes, such as necrosis of the epithelium and acinar cells, inflammatory cell infiltration, and proliferation of pancreatic ducts, are notable. b Higher magnification of pancreas from the same animal as in (a). Small ducts also show epithelial necrosis. Ducts obstructed by protein plugs or debris are seen. Peripheral tissues have undergone colliquative necrosis. c Pancreas at 7 days after ligation near the duodenum. Acinar cell atrophy and

Fig. 4. Characteristics of pancreaticobiliary duct-ligation model. a Pancreas at 3 days after ligation near the duodenum. Drastic changes, such as necrosis of the epithelium and acinar cells, inflammatory cell infiltration, and proliferation of pancreatic ducts, are notable. b Higher magnification of pancreas from the same animal as in (a). Small ducts also show epithelial necrosis. Ducts obstructed by protein plugs or debris are seen. Peripheral tissues have undergone colliquative necrosis. c Pancreas at 7 days after ligation near the duodenum. Acinar cell atrophy and

In the liver, destruction and proliferation of the bile duct, along with inflammation, were noticeable (fig. 4d). These changes were more severe in the rats with ligation closer to the duodenum than in those with ligation at the porta hepatica (fig. 4f). It was therefore considered that influx of pancreatic juice into the bile duct might affect the progression of hepatic lesions.

Pancreatitis in ALY mice

Autoimmune pancreatitis is defined as pancreatitis involving autoimmune responses [54, 55]. This type of pancreatitis is characteristically accompanied by increased levels of autoantibody and 7-globulin in the serum, and by fibrosis with lymphocytic infiltration; but clinically there is a lack of acute symptoms [51, 56, 58, 93, 94]. As an experimental model for autoimmune pancreatitis, thymectomized neonatal BALB/c mice immunized with carbonic anhydrase II or lactoferrin have been used, since these mice show T-lymphocyte-dominated infiltration into exocrine glands such as salivary glands and pancreas [55, 57, 95]. In contrast, as a model of spontaneously-occurring autoimmune pancreatitis, the alymphoplasia (ALY) mouse is well recognized [96-98]. The ALY mouse is immunodeficient, as it lacks generalized lymph nodes and Peyer's patches and, moreover, shows structural abnormalities in its spleen and thymus. It was mentioned that histopathological characteristics were chronic inflammatory changes in exocrine organs such as the salivary gland, lacrimal glands and pancreas in homozygotes (aly/aly), but not in heterozygotes (aly/+) [97, 98]. In our examinations, inflammation was located in periductal areas in the early stage (fig. 5a), fibrosis gradually appeared in an age-related manner, and there was marked proliferation of the pancreatic ducts (fig. 5b, c). Inflammatory changes were severer in the pancreas than in the salivary glands (fig. 5d) or lacrimal glands (fig. 5e). In 30-week-old aly mice, the acinar cells had become atrophic and the ductal proliferation increasingly dominant. On the other hand, the fibrosis around the pancreatic ducts did not undergo involution during the experimental period. We attribute the irreversibility of the pancreatic fibrosis in ALY mice to persistent inflammation around the pancreatic ducts.

ductal hyperplasia are widespread. Ductal proliferation is accompanied by periductal fibrosis. Inflammatory cell infiltration also has continued. d Liver at 7 days after ligation near the duodenum. Unlike in the case of ligation at the porta hepatica f), various changes are observed around Glisson's sheath. Epithelial necrosis of the bile ducts is similar to that of the pancreatic ducts. Necrosis of hepatocytes, inflammatory cell infiltration, and fibrosis can be observed in this model. This hepatic necrosis is thought to be a major difference between the DBTC and ligation models in our experiments.The latter possibly involves the influx of pancreatic juice into the bile duct. e Pancreas at 3 days after ligation at the porta hepatica. In the case of common bile duct ligation (a), no abnormalities are seen in the pancreas. f Liver at 7 days after ligation at the porta hepatica. Proliferation of bile ducts with infiltration of inflammatory cells is seen.

Fig. 5. Characteristics of spontaneously occurring autoimmune pancreatitis in ALY mice. a Pancreas from an ALY mouse at 8 weeks of age. Inflammatory cells accumulate around the pancreatic duct. Slight hemorrhage is seen. b Pancreas from an ALY mouse at 20 weeks of age. Fibrosis is formed around the pancreatic ducts. Inflammatory cell infiltration into the periductal area and proliferation of ducts are seen. c Pancreas from an ALY mouse at 30 weeks of age. Atrophy of acinar cells is notable. Inflammatory cells continue to

Fig. 5. Characteristics of spontaneously occurring autoimmune pancreatitis in ALY mice. a Pancreas from an ALY mouse at 8 weeks of age. Inflammatory cells accumulate around the pancreatic duct. Slight hemorrhage is seen. b Pancreas from an ALY mouse at 20 weeks of age. Fibrosis is formed around the pancreatic ducts. Inflammatory cell infiltration into the periductal area and proliferation of ducts are seen. c Pancreas from an ALY mouse at 30 weeks of age. Atrophy of acinar cells is notable. Inflammatory cells continue to

Pancreatitis in OLETF Rats and STZ Model

Otsuka Long Evans Tokushima Fatty (OLETF) rats are known as a strain which lacks the CCK-1 receptor and spontaneously develops diabetes, and they have been used as a model of human type II diabetes [99-101]. It was reported that infiltration of connective tissue into the islets of Langerhans occurred in OLETF rats at 20 weeks of age or later, and that thereafter the fibrosis in the islets progressed, the islets became enlarged, and fibrosis subsequently spread to the peripheral tissue [102, 103]. The islets were eventually divided into many pieces and functional disorder ensued. Also in our experience, fairly extensive fibrosis around the islets occurred with atrophy of acinar cells. In the enlarged islets or the surrounding fibrotic area, hemosiderin or inflammatory cell infiltration was often observed, and edema was noted in exocrine tissue (fig. 6a-d). However, fibrosis in this strain is also considered to be reversible, and, moreover, adipose tissue gradually replaces the areas of fibrosis.

On the other hand, streptozotocin (STZ) treatment is known to induce dysfunction of islets, and also has been used to prepare a model of type II diabetes. In our experience, the time of occurrence or degree of diabetes after treatment can vary a great deal among STZ-treated rats (intravenously injected with 60mg/kg). Also, in this model, we found that fibrosis occurred, dividing the islets, and partly extending around the islet tissue (fig. 6e, f). However, under our experimental conditions, fibrosis and atrophy of acinar cells in the STZ-model rats were weaker than in the OLETF rats.

Pancreatitis in DahlS Rats

DahlS rats were reported to be a salt-sensitive model of hypertension [104]. There have been many reports about the nephrotoxicity [105, 106] in these animals but little information about pancreatic changes [107]. Although this strain is not a major model for studying pancreatic fibrosis, fibrosis can be sporadically seen in these animals. In our experience, slight hemorrhage or degeneration and necrosis of acinar cells could be occasionally seen in lobules of DahlS rats at 10 weeks of age or older (fig. 7a, b). However, the fibrosis, which became marked as the animals aged further, was limited to periarterial areas (fig. 7c). We believe that the periarterial fibrosis in this strain may not be associated with intralobular changes, and that the characteristic fibrosis is an after-effect of hypertension.

infiltrate and fibrosis is widespread. d Parotid gland from an ALY mouse at 30 weeks of age. Inflammatory cell infiltration into the periductal area is seen. Periductal fibrosis is not as pronounced as in the pancreas. e Lacrimal gland from an ALY mouse at 30 weeks of age. Changes are at the same level as those in the parotid gland (c).

Fig. 6. Characteristics of islet fibrosis in OLETF rats and STZ-treated rats. a Pancreatic islets from an OLETF rat at 4 months of age. The islets are enlarged and divided by fibrous elements. b Pancreas from the same animal as (a). Acute pancreatitis is seen, i.e. widespread edema is noted. There is slight hemorrhage and infiltration by inflammatory cells. c Pancreatic islets from an OLETF rat at 8 months of age. Fibrosis has

Fig. 6. Characteristics of islet fibrosis in OLETF rats and STZ-treated rats. a Pancreatic islets from an OLETF rat at 4 months of age. The islets are enlarged and divided by fibrous elements. b Pancreas from the same animal as (a). Acute pancreatitis is seen, i.e. widespread edema is noted. There is slight hemorrhage and infiltration by inflammatory cells. c Pancreatic islets from an OLETF rat at 8 months of age. Fibrosis has

Fig. 7. Characteristics of pancreatic fibrosis in DahlS rats; a spontaneously occurring hypertensive model. a Pancreas from a DahlS rat at 10 weeks of age. Vacuoles are observed in acinar cells. Atrophic acinar cells are scattered. b Higher magnification of pancreas from the same animal as in (a). Hemosiderin deposited by the side of the small arteries. c Pancreas from a DahlS rat at 20 weeks of age. Apart from arterial changes such as medial necrosis, periarterial fibrosis can be seen. Also a small amount of fibrous material can be seen in the lobules.

Fig. 7. Characteristics of pancreatic fibrosis in DahlS rats; a spontaneously occurring hypertensive model. a Pancreas from a DahlS rat at 10 weeks of age. Vacuoles are observed in acinar cells. Atrophic acinar cells are scattered. b Higher magnification of pancreas from the same animal as in (a). Hemosiderin deposited by the side of the small arteries. c Pancreas from a DahlS rat at 20 weeks of age. Apart from arterial changes such as medial necrosis, periarterial fibrosis can be seen. Also a small amount of fibrous material can be seen in the lobules.

expanded around the islets. Evidence of atrophy of acinar cells and proliferation of pancreatic ducts is present. d Pancreas from the same animal as (c). Atrophy of acinar cells is notably widespread, and these cells have been displaced by adipose tissues. Fibrosis in and around islets still remains. e Pancreatic islets from a rat at 4 days after treatment with 60 mg/kg of STZ. Fibrous elements can be observed in the islets but there are fewer than in OLETF rats. Also, hemosiderin deposits are seen. f Pancreatic islets from a rat at 2 weeks after treatment with STZ. Progression of fibrosis and destruction of islets are not as advanced as in OLETF rats.

Fig. 8. Characteristics of ethionine-induced pancreatitis. a Pancreas from a control mouse. No abnormalities are seen. b Pancreas at 1 day after feeding a choline-deficient plus 0.5% ethionine diet. Swelling of acinar cells is noticeable. c Pancreas at 2 days after treatment. Vacuoles are seen in acinar cells. A scattering of necrotic acinar cells is evident. d Pancreas at 5 days after treatment. Necrosis of acinar cells has become prominent. Inter-and intralobular edema is observed. e Juxtapancreatic lymph node at 5 days after treatment.

Fig. 8. Characteristics of ethionine-induced pancreatitis. a Pancreas from a control mouse. No abnormalities are seen. b Pancreas at 1 day after feeding a choline-deficient plus 0.5% ethionine diet. Swelling of acinar cells is noticeable. c Pancreas at 2 days after treatment. Vacuoles are seen in acinar cells. A scattering of necrotic acinar cells is evident. d Pancreas at 5 days after treatment. Necrosis of acinar cells has become prominent. Inter-and intralobular edema is observed. e Juxtapancreatic lymph node at 5 days after treatment.

Ethionine-induced Pancreatitis

Ethionine, a metabolic analogue of methionine, is an inhibitor of protein synthesis and has been used for investigations on metabolic disturbances in pancreatitis [108-112]. There are some reports indicating that ethionine alone can induce pancreatitis in animals, although ethionine treatment of animals given a choline-free diet has an indisputably greater inductive effect [113, 114]. The causes of this experimental pancreatitis evoked by ethionine in the context of choline deficiency are commonly believed to be deficient secretion and intracellular activation of zymogen granules, since a marked increase in the number of zymogen granules in acinar cells in the initial stage and a decrease in pancreatic enzyme concentrations in the blood have been found in model animals [113, 115].

We performed the following experiment treating choline-deficient mice with ethionine: Five-week-old mice were given a choline-deficient diet with or without 0.5% ethionine and sacrificed at 1, 2, 3, 4 or 5 days after initiation of the treatment.Their pancreata were removed and histologically examined. One day after initiation of the treatment, the acinar cells swelled and the number of their zymogen granules increased (fig. 8b). After 2 days, fine vacuoles were noted in the acinar cells (fig. 8c). Necrosis of these cells became prominent at 5 days (fig. 8d). Although apoptotic figures were seen, self-digestion was considered as the main cause of cell death. However, fibrosis did not progress in pancreas during the experiment period. Therefore, it might be necessary to use animals following withdrawal after a certain period of ethionine treatment for investigations on chronic pancreatitis or pancreatic fibrosis.

In addition, in the juxtapancreatic lymph nodes, infiltrates of white blood cells and their necrosis were often conspicuous. These changes in lymph nodes seemed to be associated with the pancreatitis (fig. 8e). Furthermore, hepatic injury and pancreatic failure remarkably occurs in this model [116-118]. In our experiments, fatty changes and necrosis of hepatocytes were obvious even just 1 day after the start of treatment (fig. 8f). It is therefore necessary to be aware that severe hepatic failure occurs in this model even more than pancreatitis, and to consider the degree of involvement of other factors, especially those from hepatic failure, in the study of therapeutic effects on acute pancreatitis.

Edema, neutrophilic infiltration and necrotic debris are observed. f Liver at 1 day after treatment. Widespread necrosis of hepatocytes is seen. This may indicate that the treatment induced not only pancreatic failure but also undeniable hepatic injury.

Arginine-induced Pancreatitis

Arginine-induced pancreatitis is also classified as a metabolic disorder and is used in many studies to clarify the mechanism of pancreatits [119-125]. Amino acid imbalance and alteration of protein metabolism initially occur, followed by cessation of synthesis of zymogen granules and necrosis of acinar cells [121-123]. Moreover, there are reports indicating that excess arginine reduces pyrimidine biosynthesis and thus inhibits nucleic acid and protein synthesis [124]. Swelling and rounding of mitochondria or dilation of the endoplasmic reticulum are also reported to be predominant changes in pancreatic acini [125].

We did an experiment on arginine-induced pancreatitis in mice. Mice were intraperitoneally injected with L-arginine-HCl at doses of 3, 6 or 9g/kg twice, with a 1-hour interval between injections, and were sacrificed at 3, 6, 9, 12 or 24 h, or 2, 3, 7 or 14 days after the treatment. At 24 h after treatment, vacuoles of various sizes and necrosis were seen in the acinar cells (fig. 9b, c). Hypertrophy of acinar cells, which was observed in the pancreatitis induced by the ethionine plus choline-deficient diet, was not observed in the case of the ariginine-induced disease. Necrosis and inflammatory changes continued for 3 days afterward (fig. 9d). At 7 days after treatment, there was a tendency toward recovery. In any case, there was no fibrosis or ductal proliferation under our experimental conditions.

Fas Ligand or LPS-induced Pancreatitis

There are many reports that indicate a relationship between apoptosis and pancreatitis [126-133]. It is prudent to distinguish apoptosis from necrosis and to consider the involvement of fas/APO-1 ligands [134-136]. In our investigation on the appearance of apoptotic cells, detected by the terminal deoxynu-cleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method [137, 138], TUNEL-positive cells were observed in the inflamed area in the pancreas from animal models and humans. It is therefore possible that apoptosis arises in association with pancreatitis. However, we were interested in the order of events in pancreatitis, i.e. whether apoptosis was induced after inflammation or whether apoptosis induced inflammation. We therefore treated animals with Jo2, an antibody with the capability to mediate apoptosis. Hundred micrograms of Jo2/animal was intravenously injected into ICR, C3H/HeN and DBA/1J mice [139, 140]. It had been earlier confirmed that this dosage was sufficient to induce apoptosis in immune-system organs. As a result, at the stage when abundant apoptotic figures were seen in thymus, lymph nodes and spleen, apoptotic acinar cells in the pancreas were very few. Inflammatory changes were not induced in the pancreas (fig. 10a, b). These facts suggest that pancreatic acinar cells are less likely to express fas/APO-1 on their surface in comparison with immune cells and that the cells rarely go into apoptosis in the presence of

Fig. 9. Characteristics of arginine-induced pancreatitis. a Pancreas from a control mouse. No abnormalities are seen. b Pancreas at 6 h after the last treatment with 9 g/kg. Small vacuoles are seen in acinar cells. c Pancreas at 12 h after the last treatment with the same dose as (b). Comparatively large vacuoles have appeared. Scattered necrosis of acinar cells is evident. d Pancreas at 3 days after the last treatment with the same dose as (b). There is scattered colliquative necrosis. Inflammatory changes are very slight and not much different from (c).

Fig. 9. Characteristics of arginine-induced pancreatitis. a Pancreas from a control mouse. No abnormalities are seen. b Pancreas at 6 h after the last treatment with 9 g/kg. Small vacuoles are seen in acinar cells. c Pancreas at 12 h after the last treatment with the same dose as (b). Comparatively large vacuoles have appeared. Scattered necrosis of acinar cells is evident. d Pancreas at 3 days after the last treatment with the same dose as (b). There is scattered colliquative necrosis. Inflammatory changes are very slight and not much different from (c).

sufficient quantities of fas/APO-1 ligands in serum. Moreover, it might also be possible to conclude that apoptosis is not a main cause of pancreatitis. At any rate, animals treated with Jo2 died at an early date but did not show any signs of fibrosis at the time of their death.

Fig. 10. Characteristics of pancreas from animals treated with Jo2 (anti-Fas antibody) or LPS. a Pancreas from a C3H/HeN mouse at 1 h after treatment with Jo2. Only a few apoptotic cells are seen. b Pancreas from a C3H/HeN mouse at 4 h after treatment with 100 ^g of Jo2 per animal. A few apoptotic cells are seen. At the same time, many more cells showing apoptosis are seen in the liver, spleen, lymph nodes, bone marrow, intestinal epithelium, and other organs. The pancreas is therefore possibly not so sensitive to Fas-ligand. After withdrawal of the treatment, fibrosis is not observed. c Pancreas from a New Zealand White (NZW) rabbit at 4h after the last treatment with LPS. Apoptotic cells are scarce. d Pancreas from a NZW rabbit at 6h after the last treatment with LPS. Apoptotic cells have increased in number, and vacuolar degeneration is marked.

Fig. 10. Characteristics of pancreas from animals treated with Jo2 (anti-Fas antibody) or LPS. a Pancreas from a C3H/HeN mouse at 1 h after treatment with Jo2. Only a few apoptotic cells are seen. b Pancreas from a C3H/HeN mouse at 4 h after treatment with 100 ^g of Jo2 per animal. A few apoptotic cells are seen. At the same time, many more cells showing apoptosis are seen in the liver, spleen, lymph nodes, bone marrow, intestinal epithelium, and other organs. The pancreas is therefore possibly not so sensitive to Fas-ligand. After withdrawal of the treatment, fibrosis is not observed. c Pancreas from a New Zealand White (NZW) rabbit at 4h after the last treatment with LPS. Apoptotic cells are scarce. d Pancreas from a NZW rabbit at 6h after the last treatment with LPS. Apoptotic cells have increased in number, and vacuolar degeneration is marked.

On the other hand, approaches to induce pancreatitis by treatment with LPS have been attempted for a long time. There are many reports on models prepared by intraductal injection of endotoxin for the purpose of elucidating the mechanisms of infection or immunization [141-144]. We also have had experience in studying LPS-induced pancreatitis in rabbits. In brief, New Zealand White rabbits received LPS (5 |xg/kg, purified from Salmonella minnesota)

[145, 146] at 0, 5 and 24h were histopathologically investigated. At 24h after the last treatment with LPS, the rabbits showed signs of apoptosis in acinar cells (fig. 10c, d), however, the signs were more frequent in inflammatory cells than in pancreatic acinar cells. When the pancreata from the LPS-injected rabbits showed changes indicative of apoptosis, hemorrhage, thrombus formation and necrosis had already appeared in the livers, kidneys, spleens or hearts from the same rabbits. It goes without saying that these pancreatic changes would depend on the factors leading to multiple organ failure, which is commonly noted in septicemic diseases, rather than on the direct effect from LPS.

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