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Oxidative stress is an important factor in the pathogenesis of acute pancreatitis, as shown in vivo by the beneficial effects of scavenger treatment and in vitro by the potential of free radicals to induce acinar cell damage. However, it is still unclear whether oxygen free radicals (OFR) act only as mediators of tissue damage or represent the initiating event in acute pancreatitis in vivo as well. In the present study the authors aimed to address this issue in an experimental set-up.Two hundred male Wistar rats were randomly assigned to one of the following experimental groups. In two groups, acute necrotizing pancreatitis was induced by retrograde intraductal infusion of 3% sodium taurocholate. Through the abdominal aorta, a catheter was advanced to the origin of the celiac artery for continuous regional arterial (CRA) pretreatment with isotonic saline (NP-S group) or superoxide dismutase/catalase (NP-SOD/CAT group). In another group, oxidative stress was generated by CRA administration of xanthine oxidase and intravenous administration of hypoxanthine (HX/XOD group). Sham-operated rats received isotonic saline both arterially and intraductally. After observation periods of 5 and 30 minutes and 3 and 6 hours, the pancreas was removed for light microscopy and determination of reduced glutathione (GSH), oxidized glutathione (GSSG), conjugated dienes (CD), and malondialdehyde as a marker for OFR-induced lipid peroxidation as well as myeloperoxidase as a parameter for polymorphonuclear leukocyte accumulation.A significant decrease of GSH was paralleled by an increased ratio of GSSG per total glutathione and elevated CD levels after 5 minutes in the NP-S group versus the sham-operated group. Thereafter, the percentage of GSSG and GSH returned to normal levels until the 6-hour time point. After a temporary decrease after 30 minutes, CD levels increased again at 3 hours and were significantly higher at 6 hours in contrast to sham-operated rats. Myeloperoxidase levels were significantly elevated at 3 and 6 hours after pancreatitis induction. In contrast to NP-S rats, treatment with SOD/CAT significantly attenuated the changes in glutathione metabolism within the first 30 minutes and the increase of CDs after 6 hours. HX/XOD administration lead to changes in levels of GSH, GSSG, and CDs at 5 minutes as well as to increased myeloperoxidase levels at 3 hours; these changes were similar to those observed in NP-S rats. Acinar cell damage including necrosis was present after 5 minutes in both NP groups, but did not develop in HX/XOD rats. In addition, serum amylase and lipase levels did not increase in the latter group. SOD/CAT treatment significantly attenuated acinar cell damage and inflammatory infiltrate compared with NP-S animals during the later time intervals.OFRs are important mediators of tissue damage. However, extracellular OFR generation alone does not induce the typical enzymatic and morphologic changes of acute pancreatitis. Factors other than OFRs must be involved for triggering acute pancreatitis in vivo.

Using a retrograde infusion sodium taurocholate pancreatitis model in the rat treatment with oxygen radical scavengers or monoclonal anti-ICAM-1 antibody decreased tissue damage and polymorphonuclear leukocytes (PMN) infiltration. Scavengers or anti-ICAM-1 treatment attenuated the activating capacity of blood PMNs following zymosan stimulation. The local production of oxygen free radicals in the pancreas by systemic infusion of hypoxanthine and regional infusion of xanthine oxidase did not induce acute pancreatitis, although an increase of infiltrating PMNs was observed. Our data suggest that oxygen free radicals and infiltrating PMNs aggravate acute pancreatitis and that both are important mediators of local destruction and systemic activation of PMNs.

It has been suggested that endotoxin triggers translocation of intestinal bacteria in vivo, either by directly damaging intestinal mucosa or by inducing a systemic inflammatory reaction that leads to mucosal disruption. To address this issue, we examined the immediate effect of extraluminal endotoxin on structure and function of isolated rat cecal mucosa without other inflammatory cells in vitro. The cecal mucosa of 12 male Wistar rats was mounted in modified Ussing chambers filled with Dulbecco's modified Eagle's medium and the ampicillin-resistant Escherichia coli HB101:K12 incubated on the mucosal side. Endotoxin was added to the submucosal side at concentrations of 1 and 10 EU/ml, respectively. Under gassing with carbogene at 37 degreesC, the potential difference across the mucosa was measured continuously. Samples of the mucosal and submucosal solutions were removed at 60, 120, and 180 min and plated out on McConkey ampicillin-agar. After 180 min, the mucosal specimens were retrieved and examined by light and scanning electron microscopy. No significant change in potential difference was observed in control or endotoxin-incubated mucosa within the observation period. Neither light nor scanning electron microscopy showed a significant change in the structure of the epithelium, mucosa, or submucosa. No significant translocation of the E. coli across the mucosa was seen. We concluded that endotoxin alone does not induce immediate structural and functional damage to rat cecal mucosa in vitro. Therefore, it seems unlikely that a short endotoxemia alone directly triggers bacterial translocation by disrupting intestinal mucosa, but rather, entotoxin induces a local and systemic inflammatory reaction that leads to mucosal disruption.

Oxygen radicals mediate an important step in the initiation of acute pancreatitis. These reactive oxygen metabolites are generated at an early stage of the disease. The source of the enhanced production of oxygen radicals, however, still remains unclear. Experimentally, the effectiveness of antioxidant treatment varies from one model to the other, the differences depending on the experimental model and not on the form of pancreatitis that was induced. In most studies, the experimental animals were pretreated before acute pancreatitis was induced. This does not mirror clinical reality because patients are admitted to the hospital after the onset of the disease. It was shown in cerulein-induced pancreatitis, however, that scavenger treatment also mitigated the pancreatic tissue damage after induction of acute pancreatitis. Moreover, antioxidant treatment also attenuated the extrapancreatic complications, thus improving the final outcome of the disease. Initial indirect observations also suggest that in human acute, acute recurrent, and chronic pancreatitis, oxygen free radicals are generated and add to the damage. Concomitantly, these patients suffer from a severe depletion of oxidative stress. Whether or not this disbalance is instrumental in the development and course of disease remains unanswered. Supplementation with antioxidants that are deficient in patients with acute pancreatitis might be a feasible option to the present therapy to avoid extrapancreatic complications. Well-defined, controlled clinical trials involving patients suffering from acute pancreatitis are therefore needed to validate the role of oxygen radicals in this disease.

Hemorrhagic mucosal lesions are produced during intestinal ischemia and after reperfusion due at least in part to the accumulation and activation of polymorphonuclear leukocytes in the tissue. It has been shown in vitro that adenosine is instrumental in attenuating this pathophysiological process. Acadesine [5-amino-4-imidazolecarboxamide (AICA) riboside], a purine nucleoside analogue, increases the availability of adenosine in the tissue. The aim of the study was therefore to assess the influence of acadesine treatment on neutrophil accumulation, purine metabolism, and mucosal damage after intestinal ischemia and reperfusion. Intestinal ischemia was induced in cats by partial occlusion of the superior mesenteric artery for 2 h. Samples of the small intestine were exercised before and at the end of the hypotensive period as well as 10 and 60 min after reperfusion. Conjugated dienes, myeloperoxidase, and reduced and oxidized glutathione, as well as the purine metabolites, were determined in the tissue samples. The tissue was also examined histologically. Six cats received saline, and six cats were treated initially before ischemia with acadesine (2.5 mg/kg body wt i.v.) over 5 min as a bolus. Thereafter, acadesine (0.5 mg.kg-1.min- i.v.) was given continuously during ischemia and 30 min after reperfusion. Acadesine treatment significantly attenuated the mucosal lesions seen during reperfusion. This improvement was due at least in part to the inhibition of neutrophil accumulation, as judged by low myeloperoxidase levels. The prevention of neutrophil activation resulted most likely from increased adenosine concentrations in the intestinal tissue in the early reperfusion period.(ABSTRACT TRUNCATED AT 250 WORDS)

Background/Aim : In spite of the interest in free radicals as mediators of ischemic damage, most information on these species in biological systems is derived from indirect measurements. Our aim was to obtain more direct information concerning sources of free radical production during ischemia and reperfusion. Methods : We have performed simultaneous measurement of radical generation, purine metabolites, reduced glutathione, neutrophil infiltration and morphological appearance in the cat small intestine in vivo during 60 minutes of ischemia followed by 60 minutes of reperfusion. Results : Radical formation increased abruptly on reperfusion and remained elevated in untreated animals. Inhibition by a monoclonal antibody (IB4) against the neutrophil and by allopurinol treatment was paralleled by improvement of biochemical and morphological parameters. The radicals detected during reperfusion could be divided into one component arising directly from the neutrophils, one due to the xanthine oxidase reaction, and one unknown source. Conclusions : Neutrophils are a major source of radical production during reperfusion after ischemia. Radicals formed in the xanthine oxidase reaction seem to function as a primer for the neutrophils. The nonsignificant linear correlation between radical formation and morphological appearance suggests that factors other than free radicals are important for the development of intestinal damage after a period of ischemia.

Review the histologic and pathophysiologic alterations seen after intestinal ischemia and reperfusion.Current literature review.The most pertinent, current, and representative articles describing results from both animal and human investigations are utilized and discussed.Postischemic intestinal tissue damage appears to be due to the formation of oxygen radicals and the activation of phospholipase A2. The initial source of oxygen radicals seems to be the hypoxanthine-xanthine oxidase system. Oxygen radicals react directly with poly-unsaturated fatty acids, leading to lipid peroxidation within the cell membranes. Indirectly, the radicals trigger the accumulation of neutrophils within the affected tissue initiating inflammatory processes that lead to severe mucosal lesions. Similarly, phospholipase A2 also initiates postischemic mucosal lesions. Phospholipase A2 is a hydrolytic enzyme capable of increasing formation of cytotoxic lysophospholipids within the tissue. Enhanced activity of phospholipase A2 also stimulates the production of prostaglandins and leukotrienes. Various substances (superoxide dismutase, catalase, dimethyl sulfoxide, allopurinol, and deferoxamine, etc.) are able to detoxify oxygen radicals or inhibit the mechanisms leading to their enhanced generation, thus attenuating the postischemic lesions of the mucosa.Oxygen radicals and the activation of phospholipase A2 during reperfusion seem to be instrumental for the development of hemorrhagic mucosal lesions after intestinal ischemia. Radical scavengers and phospholipase A2 inhibitors may prevent reperfusion damage of the intestine, even when the treatment starts during ischemia but before reperfusion.

The purpose of this study was to assess the involvement of oxygen radicals in acute edematous and hemorrhagic panreatitis. Acute pancreatitis was induced in rats by the CCK-analogue cerulein (5 μg/kg/h) and by retrograde injection of 5% sodium taurocholate for 30 min, 3.5 h, and 12 h. At the end of the infusion and observation time, serum enzymes, conjugated dienes, and malondialdehyde in the tissue were measured. Moreover, the tissue samples underwent light microscopical examination. In cerulein pancreatitis, an interstitial edema and intravascular margination of granulocytes in the pancreatic gland were observed after 3.5 h. After 12 h, the histological evaluation revealed a pronounced zymogen degranulation, extensive tissue necrosis and migration of granulocytes into the tissue. Parallelly, amylase and lipase increased by 15 and 35 times, respectively. In contrast, conjugated dienes and malondialdehyde increased in cerulein pancreatitis and reached their highest level after 3.5 h and decreased to normal levels after 12 h. The development of the histological damages and serum enzyme levels with sodium taurocholate pancreatitis was similar as compared to the cerulein pancreatitis, however, the development was faster and more traumatic. Already after 3.5 h an extensive zymogen degranulation and cell necrosis was observed. Concomitantly, the amylase and lipase levels increased by 90 and 30 times, respectively. Treatment with superoxide dismutase (100000 U/kg/h) and catalase (400 000 U/kg/h) prevented lipid peroxidation and reduced zymogen degranulation and tissue necrosis. Tissue edema and inflammatory response were not affected in both models of acute pancreatitis. Moreover, scavenger treatment improved the survival rate in sodium taurocholate pancreatitis. It is therefore concluded that oxygen free radicals seem to be instrumental for the development of the disease. Scavenger treatment can mitigate the pancreatic tissue damages and the extrapancreatic complications.

Background & Aims Pancreatic cancers contain exclusively tumorigenic cancer stem cells (CSCs), which are highly resistant to chemotherapy, resulting in a relative increase in CSC numbers during gemcitabine treatment. Signaling through sonic hedgehog and mammalian target of rapamycin (mTOR), respectively, may be essential for CSC self-renewal and could represent putative targets for novel treatment modalities. Methods We used in vitro and in vivo models of pancreatic cancer to examine the effects of sonic hedgehog inhibition (cyclopamine/CUR199691) and mTOR blockade (rapamycin) on the tumorigenic CSC population. Results Surprisingly, neither cyclopamine nor rapamycin alone or as supplements to chemotherapy were capable of effectively diminishing the CSC pool. Only the combined inhibition of both pathways together with chemotherapy reduced the number of CSCs to virtually undetectable levels in vitro and in vivo. Most importantly, in vivo administration of this triple combination in mice with established patient-derived pancreatic tumors was reasonably tolerated and translated into significantly prolonged long-term survival. Conclusions The combined blockade of sonic hedgehog and mTOR signaling together with standard chemotherapy is capable of eliminating pancreatic CSCs. Further preclinical investigation of this promising approach may lead to the development of a novel therapeutic strategy to improve the devastating prognosis of patients with pancreatic cancer.

Pancreatic hyperstimulation with simultaneous duct obstruction does not cause the typical features of acute pancreatitis, therefore the role of an additional challenge, such as either ethanol intoxication or short-term ischemia, was studied. Alcoholic pancreatitis was induced in 28 rats by acute ethanol intoxication (0.25 LD 50 ) and an obstruction/ hyperstimulation mechanism (clip of the biliopancreatic duct for 20 min and intravenous stimulation with 5 U of cholecystokinin and secretin each). Ischemic pancreatitis was performed by obstruction/hyperstimulation and subsequent pancreatic ischemia by clamping the supplying arteries for 40 min. The macro- and microscopic alterations were evaluated and graded by a scoring system. Additionally, the pancreas was removed in 50% of the animals and the pancreatic acini were prepared. From those acini the intracellular enzymes trypsinogen, kallikreinogen, amylase, lipase, glucuronidase, and acidic phosphatases were determined. While obstruction/hyperstimulation, 40 min of ischemia, or ethanol alone did not induce acute pancreatitis, a combination of obstruction/hyperstimulation with either ethanol or ischemia resulted in acute pancreatitis in 68 and 60% of treated rats, respectively. Similarly, both models were characterized by extrapancreatic fat necrosis and acinar necrosis at the periphery of the lobules. Almost all intracellular enzymes were elevated in both pancreatitis models compared to sham-operated controls. Both alcohol and ischemia were insults that sensitize the pancreas to develop acute pancreatitis after obstruction/hyperstimulation. Since the observed morphologic and enzymatic alterations in both models are very similar, alcohol and ischemia might have some common pathways by which they make the pancreas vulnerable to enzymatic attacks.

The purpose of this study was to evaluate the effect of free radical ablation therapy in acute hemorrhagic pancreatitis. Acute pancreatitis was induced in 64 rats by retrograde injection of 5% sodium taurocholate. Thirty animals were pretreated with 100,000 units/kg/hr of superoxide dismutase (SOD) and 400,000 units/kg catalase within the first 3 hr. After 0.5, 3.5, and 12 hr of observation time, serum enzymes and the tissue content of conjugated dienes, malondialdehyde, reduced and oxidized glutathione, as well as ATP, ADP and AMP were measured. In addition, tissue samples were examined by light microscopy. Untreated rats (N = 34) developed within 12 hr an acute hemorrhagic necrotizing pancreatitis with a concomitant increase in serum enzyme levels and a decrease in reduced glutathione and ATP. Within the 12-hr observation period, 57% of the animals died. Scavenger treatment improved the tissue damage and attenuated the increase of the serum enzyme levels and the decrease in reduced glutathione and ATP. Moreover, the lethality rate was significantly lower. Oxygen radicals seem to be instrumental for the development of acute hemorrhagic pancreatitis. Thereby, antioxidant treatment reduces tissue damage, biochemical alterations and extrapancreatic complications, thus improving the final outcome.

A growing body of evidence suggests that oxygen radicals are generated in all forms of experimental pancreatitis at an early stage of disease. Moreover, first indirect observations assume that also in human acute recurrent and chronic pancreatitis oxygen free radicals are generated and add to the damages seen. The source of the enhanced production of oxygen radicals remains still unclear. Experimentally, the efficiency of scavenger treatment varies between three different models, whereby these differences depend more on the design of the experimental models than on the form of pancreatitis which was induced. Antioxidant treatment with radical scavengers should therefore interrupt these deleterious pathomechanisms or at least mitigate the damages normally seen. Most studies, however, pretreated the experimental animals before inducing acute pancreatitis, which does not mirror the clinical reality. Patients, however, are admitted after onset of the disease. Therefore, well-defined, controlled clinical studies are needed to validate the involvement of oxygen radicals in acute and chronic pancreatitis and the effect of scavenger treatment in patients with pancreatitis.

Oxygen-derived free radicals mediate an important step in the initiation of experimental acute pancreatitis. Thereby, it seems that these reactive oxygen metabolites are generated at an early stage of disease. The source of the enhanced production of oxygen radicals still remains unclear. Experimentally, the efficiency of scavenger treatment varied between different models, whereby these differences depended on the experimental model and not on the form of pancreatitis which was induced. Most studies pretreated the experimental animals before inducing acute pancreatitis. This does not mirror the clinical reality, since patients are admitted to the hospital after onset of the disease. It was shown in Cerulein pancreatitis, however, that scavenger treatment also mitigated the pancreatic tissue damages after induction of acute pancreatitis. Moreover, antioxidant treatment also attenuated the extrapancreatic complications, thus improving the final outcome of the disease. The first indirect observations also suggest that in human acute recurrent and chronic pancreatitis, oxygen free radicals are generated and add to the damages seen. Therefore, well-defined controlled clinical studies with patients suffering from acute pancreatitis are needed to validate the role of oxygen radicals in this disease.

The generation of free oxygen radicals is presumed as a substantial pathogenetic principle in reperfusion injury. Although demonstrated in gut, muscle and kidneys its role in liver reperfusion injury is still under investigation. In an experimental rat model of warm liver ischemia of 60 min and 8 h reperfusion electron resonance spectroscopy assessed the increased generation of free radicals in early reperfusion period, leading to a decrease of polyunsaturated free fatty acids in liver tissue within 15 min of reperfusion. Histologically, single cell death, local and patchy necrosis of hepatic lobuli could be observed after 8 h reperfusion (n = 6). These histologic signs of liver injury could be attenuated by administration of superoxide-dismutase in combination with catalase but not by allopurinol. Best results could be obtained by deferoxamine. This indicates that increased generation of free oxygen radicals in reperfusion is not caused by the known conversion of xanthine-dehydrogenase to -oxidase but is mediated by an increased generation of hydroxyl-radicals, which can be scavenged by deferoxamine.

Haemorrhagic mucosal lesions are produced during intestinal ischaemia and after reperfusion probably mediated by oxygen radicals. Oxygen radicals react with cell membrane lipids and induce cell damage by peroxidation and induce accumulation of polymorphonuclear leucocytes in the tissue. The aim of the study was to elucidate the involvement of polymorphonuclear leucocytes in post-ischaemic intestinal damage. Intestinal ischaemia was induced in cats by partial occlusion of the superior mesenteric artery. Samples from the small intestine were excised before and at the end of the two hour hypotensive period as well as 10 minutes and 60 minutes after reperfusion. Conjugated dienes, myeloperoxidase, and the purine metabolites were determined in the samples. The tissue was also examined histologically. Seven cats were treated before reperfusion with a monoclonal antibody (IB4) which inhibits leucocyte adherence to endothelial cells and its subsequent activation. After reperfusion myeloperoxidase activity increased and the ischaemic mucosal lesions worsened significantly. IB4 treatment prevented an increase in post-hypotensive myeloperoxidase activity and attenuated the normally observed severe mucosal lesions. We conclude that the severe post-ischaemic lesions are induced by polymorphonuclear leucocytes. Such mucosal injury may be appreciably reduced by blocking leucocyte adherence with IB4.

This study aimed to assess the role of oxygen free radicals in acute pancreatitis. Acute pancreatitis was induced in rats by infusion of the CCK-analogue cerulein (5 micrograms/kg per hour) for 30 minutes, 3.5 hours, and 12 hours. After the infusion, serum enzymes and conjugated tissue dienes and malondialdehyde were measured and tissue samples were subjected to electron and light microscopy. Electron microscopy after 30 minutes showed moderate intracellular alterations. After 3.5 hours of cerulein infusion interstitial oedema and intravascular margination of granulocytes in the pancreatic gland were seen. After 12 hours histological evaluation showed pronounced zymogen degranulation, extensive tissue necrosis, and migration of granulocytes into the tissue. Amylase and lipase activities increased 15 and 35-fold respectively during this time. After 30 minutes of cerulein infusion conjugated dienes and malondialdehyde increased, they reached their peak after 3.5 hours and decreased to normal values after 12 hours. Treatment with superoxide dismutase (100,000 U/kg/hour) and catalase (400,000 U/kg/hour) either before or after the start of the cerulein infusion prevented lipid peroxidation and reduced zymogen degranulation and tissue necrosis. Tissue oedema and inflammatory response, however, were not affected in any of the treated rats. Oxygen free radicals are instrumental in the development of acute pancreatitis. Even after its onset, scavenger treatment reduced the tissue damage normally observed.

Characteristic mucosal lesions develop in the small intestine during ischaemia and hypotension. This tissue damage can be further aggravated in the immediate reperfusion phase, presumably secondary to the generation of oxygen free radicals which have been proposed to be generated in this situation through the hypoxanthine-xanthine oxidase system. This was further investigated in the cat small intestine using a standardized regional intestinal hypotension model in which the effects of allopurinol (a xanthine oxidase inhibitor) were compared to those of an exogenous supply of inosine. The grade of mucosal damage, the nucleotide levels, the concentrations of hypoxanthine, total and oxidized glutathione, and of conjugated dienes were measured in the intestinal tissue. The results indicate that oxygen radicals generated by xanthine oxidase are very important, but not the only significant factor in the small intestinal reperfusion damage.

The purpose of this study was to assess the involvement of oxygen radicals and phospholipase A2 in acute pancreatitis. Acute pancreatitis was induced in rats by the CCK-analogue cerulein (5 micrograms/kg.h) for 30 min, 3.5 h, and 12 h. At the end of the infusion, serum enzymes and the lipid peroxidation products conjugated dienes and malondialdehyde in the tissue were measured. Moreover, tissue samples underwent lightmicroscopical examination. After 3.5 h cerulein, an interstitial edema and a beginning accumulation of granulocytes in the pancreatic gland is observed. These changes are aggravating within 12 h, leading to tissue necrosis and migration of the granulocytes into the tissue. Concomitantly amylase and lipase increased by 15 and 35 times, respectively. Conjugated dienes and malondialdehyde increase already after 30 min cerulein and reach their highest levels after 3.5 h cerulein. At the same time the tissue activity of phospholipase A2 is elevated three fold. Rats were treated with superoxide dismutase and catalase before cerulein infusion. Treatment significantly prevents tissue necrosis, granulocyte accumulation, and edema formation. The enhanced activity of phospholipase A2, however, is unaffected by the treatment. Oxygen radicals seem to be instrumental in the development of the disease.

The hemodynamic effectiveness and the oxygen characteristics of stroma-free hemoglobin (SFH) solutions were studied in 11 dogs. the animals were bled two-thirds of the estimated blood volume or until cardiac arrest. The shed blood was immediately replaced by equivalent amounts of either SFH or polyhemoglobin (SFH-PLP). Sixty min later, Ringer's lactate was given iv for 180 min to maintain right atrial pressure at the initial value. In a second set of experiments, the intravascular persistence of SFH-PLP was investigated in 5 dogs by withdrawal of blood (7 ml/kg bw) and immediate replacement with 131I-labeled SFH-PLP. Hemodynamic disorders after severe blood loss could be reversed by infusion of hemoglobin solutions. Due to the short intravascular persistence, cardiac output did not increase as one would expect from the degree of hemodilution. Pyridoxalation of the hemoglobin molecule reduced oxygen affinity and improved oxygen unloading at the tissue level. The short intravascular half-time of this compound could be overcome by crosslinking of the pyridoxalated hemoglobin molecules. Further studies must prove whether this polyhemoglobin can be used as a long-term oxygen-carrying blood substitute.