Intraductal Infusion Research Articles

Transient Stasis of Pancreatic Fluid Flow Together with Mild Injury of the Pancreatic Duct Cause Chronic Pancreatitis

Little is known about the etiopathogenesis of chronic pancreatitis, due mainly to the lack of simple animal models suitable to study inflammatory and fibrogenetic processes in the pancreas. The purpose of this study was to examine whether transient congestion of pancreatic fluid flow alone or slight ductal injury alone is sufficient, or where both are required, to induce chronic pancreatic injury. Three different models of pancreatitis were tested in rats induced by retrograde intraductal infusion of 40μl/100g body weight of 0.01% agarose (group A), 40μl/100g body weight of 0.1% sodium taurocholate (group T), or a mixture of the two solutions (group M). Histological alterations of the pancreas were examined by hematoxylin-eosin staining, changes in type IV collagen structure were studied by immunostaining, and the gelatinolytic activity of latent and active matrix metalloproteinase-2 (MMP-2) was analyzed by zymography. In group A and T rats, histological alterations of the pancreas and the gelatinolytic activity of MMP-2 returned to baseline levels by day 14, and immunoreactivity for type IV collagen appeared as continuous lines along the basement membrane. In group M rats, however, acinar damage, fibrosis and fatty degeneration were observed even on day 56, and type IV collagen was detected as discontinuous lines until day 56. MMP-2 was significantly elevated from day 5 to day 42. Co-existence of transient stasis of pancreatic fluid flow, together with mild damage to the pancreatic duct and acinar cells, exert synergistic effects on the development of persistent pancreatic injury with continuous disorganization of type IV collagen in the basement membrane of the ducts.

Activation of lung macrophage subpopulations in experimental acute pancreatitis

Pulmonary macrophages exist in two different anatomical compartments in the lower respiratory tract: alveolar macrophages in the alveoli and interstitial macrophages in the interstitium. Depending on the micro-environmental stimulation, macrophages follow different activation pathways. According to their inflammatory response pattern, activated macrophages have been characterized as pro-inflammatory (M1), wound-healing (M2a) and regulatory (M2b). Since acute pancreatitis occurs in parallel with acute lung injury, the profile of the different macrophage subpopulations could be relevant in the progression of the disease. The activation of lung alveolar and interstitial macrophages was assessed in an experimental model of severe acute pancreatitis induced in rats by intraductal infusion of 3.5% sodium taurocholate. Alveolar and interstitial macrophages were obtained and the expression of markers of different activations was evaluated. Activation of nuclear factors PPARγ and NF-κB, which are involved in the acquisition of different phenoytpes, was also measured. Alveolar macrophages acquired an early M1 phenotype characterized by the expression of inflammatory cytokines and NF-κB activation. In contrast, interstitial macrophages followed the inhibitory M2b pathway. In these macrophages, PPARγ became activated and the anti-inflammatory cytokine IL-10 was expressed. These results suggest that alveolar and interstitial macrophages play different roles in acute lung injury associated with acute pancreatitis. Alveolar macrophages promote an early inflammatory response, whereas interstitial macrophages help resolve inflammation.

Efeitos do cloreto de gadolínio na pancreatite induzida por tauracolato de sódio em ratos

To evaluate the effects of the use of gadolinium chloride before and after induction of acute pancreatitis with sodium taurocholate 3% in rats. Wistar rats were divided into five groups: SF--control with saline intra-ductal and IV; GD control with saline intra-ductal and gadolinium chloride IV; TS--with AP control induced by sodium taurocholate 3% and saline IV; GDTS--pre-treatment with GD (24 hours before the induction of AP) and TSGD--treatment with GD (1 hour after the induction of AP). Analysis was made in serum amylase, transaminases and TNF-α; determination of the MPO activity in lung tissue, lung and pancreatic histology. The number of dead animals before the end of the experiment was significantly higher in TSGD (P = 0.046). The scores of pancreatitis and lung damage were higher in the groups that used sodium taurocholate compared to groups with intra-ductal infusion of saline solution. There were no differences in other variables studied when comparing TS, GDTS and TSGD groups. The benefits with the use of gadolinium chloride as a prophylactic and therapeutic drug were not demonstrated.

Status of Intraductal Therapy for Ductal Carcinoma in Situ.

The intraductal approach is particularly appealing in the setting of ductal carcinoma in situ (DCIS), a preinvasive breast neoplasm that is thought to be entirely intraductal in its extent. Based on an emerging understanding of the anatomy of the ductal system as well as novel techniques to leverage the access accorded by the intraductal approach, researchers are actively exploring how ductal lavage, ductoscopy, and intraductal infusion of therapeutic agents may enhance breast cancer treatment. Both cytologic and molecular diagnostics continue to improve, and work is ongoing to identify the most effective diagnostic biomarkers for DCIS and cancer, although optimal targeting of the diseased duct remains an important consideration. Ductoscopy holds potential in detection of occult intraductal lesions, and ductoscopically guided lumpectomy could increase the likelihood of a more comprehensive surgical excision. Exciting pilot studies are in progress to determine the safety and feasibility of intraductal chemotherapy infusion. These studies are an important starting point for future investigations of intraductal ablative therapy for DCIS, because as our knowledge and techniques evolve, it is likely that DCIS may be the target most amenable to treatment by intraductal therapy. If such studies are successful, these approaches will allow an important and meaningful transformation in treatment options for women diagnosed with DCIS.

Animal Models of Chronic Pancreatitis

Animal models for CP in rats can be classified into 2 groups: one is noninvasive or nonsurgical models and the other is invasive or surgical models. Pancreatic injury induced by repetitive injections of supramaximal stimulatory dose of caerulein (Cn) or by intraductal infusion of sodium taurocholate (NaTc) recovered within 14 days, whereas that caused by repetitive injection of arginine or by intraductal infusion of oleic acid was persistent. However, the destroyed acinar tissues were replaced by fatty tissues without fibrosis. Transient stasis of pancreatic fluid flow by 0.01% agarose and minimum injury of the pancreatic duct by 0.1% NaTc solution induced progressive pancreatic injury although one alone is insufficient to cause persistent pancreatic injury. However, the damaged tissue was replaced by fatty tissue without fibrosis. Continuous pancreatic ductal hypertension (PDH) caused diffuse interlobular and intralobular fibrosis closely resembling human CP.

Pancreatic and pulmonary mast cells activation during experimental acute pancreatitis

To study the activation of pancreatic and pulmonary mast cells and the effect of mast cell inhibition on the activation of peritoneal and alveolar macrophages during acute pancreatitis. Pancreatitis was induced by intraductal infusion of 5% sodium taurodeoxycholate in rats. The mast cell inhibitor cromolyn was administered intraperitoneally (i.p.) 30 min before pancreatitis induction. The pancreatic and pulmonary tissue damage was evaluated histologically and mast cells and their state of activation were evaluated. Peritoneal and alveolar macrophages were obtained and the expression of tumor necrosis factor alpha was determined. Myeloperoxidase activity was measured to evaluate the effect of mast cell inhibition on the progression of the inflammatory process. Finally, the effect of plasma on cultured mast cells or macrophages was evaluated in vitro. The mast cell stabilizer significantly reduced inflammation in the pancreas and lung and the activation of alveolar macrophages but had no effect on peritoneal macrophages. Mast cell degranulation was observed in the pancreas during pancreatitis but no changes were observed in the lung. Plasma from rats with pancreatitis could activate alveolar macrophages but did not induce degranulation of mast cells in vitro. Pancreatic mast cells play an important role in triggering the local and systemic inflammatory response in the early stages of acute pancreatitis. In contrast, lung mast cells are not directly involved in the inflammatory response related to pancreatic damage.

Proposed protective mechanism of the pancreas in the rat

BackgroundHeparan sulphate is known to have various functions in the animal body, including surveillance of tissue integrity. Administered intraperitoneally, it induces a systemic inflammatory response syndrome and when given locally in the pancreas it initiates a protective inflammatory response. The aim of the present study was to investigate the underlying mechanisms behind cell recruitment following intra-ductal infusion of heparan sulphate.MethodsRats were subjected to intraductal-infusion of heparan sulphate, lipopolysaccharide and phosphate buffered saline into the pancreas. Pancreatic tissue was harvested 1, 3, 6, 9 or 48 hours after infusion and stained immunohistochemically for myeloperoxidase, ED-1, CINC-1 and MCP-1, as well as using eosin hematoxylin staining. Furthermore, MPO activity and MCP-1 and CINC-1 concentrations of tissue homogenates were measured. All differences were analyzed statistically using the Mann-Whitney U-test.ResultsDuring HS infusion, a rapid influx of macrophages/monocytes, as visualized as ED-1 positive cells, was seen reaching a maximum at 6 hours. After 48 hours, the same levels of ED-1 positive cells were noted in the pancreatic tissue, but with different location and morphology. Increased neutrophil numbers of heparan sulphate treated animals compared to control could be detected only 9 hours after infusion. The number of neutrophils was lower than the number of ED-1 positive cells. On the contrary, LPS infusion caused increased neutrophil numbers to a larger extent than heparan sulphate. Furthermore, this accumulation of neutrophils preceded the infiltration of ED-1 positive cells. Chemokine expression correlates very well to the cell infiltrate. MCP-1 was evident in the ductal cells of both groups early on. MCP-1 preceded monocyte infiltration in both groups, while the CINC-1 increase was only noticeable in the LPS group.ConclusionsOur data suggest that heparan and LPS both induce host defense reactions, though by using different mechanisms of cell-recruitment. This implies that the etiology of pancreatic inflammation may influence how the subsequent events will develop.

Hepatic Effects of Thoracic Epidural Analgesia in Experimental Severe Acute Pancreatitis

Thoracic epidural anesthesia (TEA) protects the intestinal microcirculation and improves perioperative outcomes. TEA also reduces mortality in acute experimental pancreatitis. Its impact on hepatic microcirculation, however, in health and critical illness is unknown. Therefore, the authors studied the effect of TEA on the liver in healthy rats and in experimental severe acute pancreatitis. TEA was induced by 15 microl/h bupivacaine, 0.5%. Necrotizing pancreatitis was induced by intraductal infusion of 2 ml/kg taurocholic acid, 5%. Twenty-eight rats were assigned to either Sham operation, Sham + TEA, Pancreatitis, or Pancreatitis + TEA. After 15 h, mean arterial pressure, heart rate, and respiratory function were recorded. Sinusoidal width and perfusion rate and the intrahepatic leukocyte adhesion were assessed by intravital microscopy. In an additional 22 rats randomly assigned to Sham, Pancreatitis, and Pancreatitis + TEA, hepatic apoptosis was evaluated by staining for single-stranded DNA and Fas ligand-positive cells. TEA did not affect hepatic microcirculation and leukocyte adhesion in healthy rats. Blood pressure remained unchanged in the Sham + TEA group. In Pancreatitis, mean arterial pressure decreased from 141 + or - 6 mmHg to 127 + or - 13 mmHg but remained stable in Pancreatitis + TEA. The sinusoidal diameter decreased from 5.4 + or - 0.1 microm to 5.0 + or - 0.2 microm in Pancreatitis. This was restored in Pancreatitis + TEA. Intrahepatic leukocyte adhesion was not affected by TEA. The increased hepatocyte apoptosis in Pancreatitis was abolished in Pancreatitis + TEA. This might be mediated by inhibition of the Fas ligand pathway. TEA reduces liver injury in necrotizing acute pancreatitis. This could be related to a regional sympathetic block. TEA could thus preserve liver function in systemic inflammatory disorders such as acute pancreatitis.

Antiapoptotic Effect of Tacrolimus on Cytokine-Challenged Human Islets

Our goal was to investigate whether previously related antiapoptotic and anti-inflammatory effects of tacrolimus could be useful in protecting human islets cultured in the presence of several proinflammatory mediators. Human islets obtained from cadaveric donors after intraductal infusion with collagenase, mechanical digestion, and continuous Ficoll gradient purification were cultured in RPMI-1640 medium for 24 h. Escherichia coli lipopolysaccharide (10 microg/ml) or interleukin-1 (50 UI/ml) + gamma-IF (1000 UI/ml) and low-dose tacroliumus (5 ng/ml) were added. Homogenized samples (300 IE) from five different donors where assigned to four different experimental groups (control, treatment, cytokines, and cytokines + treatment). To evaluate islet damage and apoptotic response, nucleosome content, Bcl-2 protein levels, caspase-3, -8, and -9 levels, and insulin concentration were measured. Also, TNF-alpha and IL-6 levels where assessed as indicators of the inflammatory response. All proapoptotic markers, TNF-alpha, and IL-6 levels were augmented after both LPS and cytokine stimulation. Tacrolimus reduced significantly all of them and restored baseline values of nucleosome and caspase-9 in both experiments and Bcl-2 and caspase-3 when IL-1 + gamma-IF was added. Twenty-four-hour insulin concentration diminished when LPS or IL-1 + gamma-IF were present. Tacrolimus treatment restored insulin levels in both experiments. These results suggest that in vitro apoptotic events and media insulin concentration decrease after proinflammatory stimulation can be reverted using low-dose tacrolimus.

Early Inhibition of Prostaglandin Synthesis by n-3 Fatty Acids Determinates Histologic Severity of Necrotizing Pancreatitis

Previously, we observed decreased histopathological severity of acute necrotizing pancreatitis (ANP) by parenteral nutrition with n-3 fatty acids. Thus, we now sequentially analyzed the impact of n-3 fatty acids on prostaglandin and leukotriene synthesis in ANP. One hundred ninety-eight Sprague-Dawley rats (11 groups, n = 18) underwent intraductal glycodesoxycholat instillation and 6-hour cerulein infusion. Afterward, saline was infused in groups 2, 4, 6, 8, and 10, whereas groups 3, 5, 7, 9, and 11 received infusion rich in n-3 fatty acids (Omegaven, Fresenius Kabi, Bad Homburg, Germany). Animals were killed after 6 (group 1), 10 (groups 2 and 3), 14 (groups 4 and 5), 18 (groups 6 and 7), 22 (groups 8 and 9), and 26 hours (groups 10 and 11). The pancreas was histopathologically examined, and the pancreatic eicosanoid metabolism (prostaglandin E2, prostaglandin F1alpha [PGF1alpha], and leukotrienes) and lipid peroxidation (thiobarbituric acid-reactive substance, superoxide dismutase, and glutathione peroxidase) were analyzed. Between the 14th and 26th hours, histopathologic scores (edema, inflammation, bleeding, and necrosis) were reduced in the n-3 fatty acid group compared with the corresponding saline group. Pancreatic prostaglandin E2 and PGF1alpha were decreased between the 10th and 18th hour by n-3 fatty acids; PGF1alpha was reduced after 26 hours compared with the corresponding saline group. Lipid peroxidation was decreased by n-3 fatty acids after 14 hours (thiobarbituric acid-reactive substance); however, there was no difference concerning lipid peroxidation protective enzymes (glutathione peroxidase and superoxide dismutase). Parenteral therapy with n-3 fatty acids decreased histopathologic severity in ANP by early inhibition of prostaglandin (E2 and F1alpha) synthesis and reduction of lipid peroxidation.

Alcohol Pretreatment Increases Hepatic and Pulmonary Injury in Experimental Pancreatitis

Background: Systemic complications including pancreatitis-associated lung injury (PALI) are critical factors that determine the outcome of severe necrotizing pancreatitis (SNP). The aim of the present study was to evaluate the role of chronicalcohol exposure on the development of PALI. Methods: 48 rats were fed either a Lieber deCarli control or alcohol diet for 6 weeks. After completion, SNP was induced by intraductal infusion of bile salt followed by intravenous infusion of cerulein over 6 h. Control animals received i.v. Ringer's solution. Intravital microscopy of the liver was performed 6 h after induction of SNP to evaluate hepatic perfusion and leukocyte adhesion. Serum parameters, edema, inflammation, and histological changes were evaluated at 12 h. IL-6 levels were evaluated in portal venous and systemic blood as well as in pancreatic tissue homogenates. Results: Alcohol pretreatment did not affect pancreatic injury in SNP. PALI was aggravated after alcohol ingestion. These animals showed increased hepatic microcirculatory disturbances, compared to SNP alone. IL-6 showed peak levels in SNP with alcohol pretreatment, although they were also elevated in SNP alone. Systemic levels of IL-6 were higher than in the portal vein. Conclusion: In SNP, alcoholic pretreatment increases pulmonary damage, while pancreatic injury is identical. The liver seems to participate in this effect by increased hepatic cytokine release.

GS19 *THE ANTIOXIDANT STATUS OF AN INFLAMMATORY DISEASE AS MEASURED BY CYCLIC VOLTAMMETRY

Objective: To determine whether serum antioxidant capacity measured by the electrochemical technique of cyclic voltammetry (CV) could resolve differences in severity of the inflammatory disease acute pancreatitis.Methods: Experimental animal study using male Wistar rats. Group 1 (baseline), immediate euthanasia with no surgical interventions; Group 2 (sham), identical surgical procedures to the pancreatitis groups but no intraductal infusion; Groups 3–5, acute pancreatitis induced by intraductal infusion of 3%, 4% or 5% sodium taurocholate respectively. Terminal blood and pancreatic tissue samples for CV, biochemistry and histology were collected at 12 h (Groups 2–5).Results: There was a significant positive correlation of the first serum cyclic voltametric peak current maxima (CVi1) with pancreatic histological severity (Spearman r = 0.51, p = 0.007) as well as with a number of other markers of systemic disease severity, notably bicarbonate (r =−0.57, p = 0.002), base excess (r =−0.65, p < 0.001) urea (r =−0.68, p < 0.001) and calcium (r =−0.60, p = 0.008). CVi1 was superior at indicating the severity of the disease state compared to a standard method of total antioxidant capacity measurement.Conclusions: CV was found to be a simple, technique that could differentiate between the physiological and histological severities of acute pancreatitis. Further clinical studies are now justified to determine if point‐of‐care CV offers prognostic and treatment‐monitoring advantages for severe acute inflammatory illnesses within the critical care setting.

Probiotics Prevent Intestinal Barrier Dysfunction in Acute Pancreatitis in Rats via Induction of Ileal Mucosal Glutathione Biosynthesis

BackgroundDuring acute pancreatitis (AP), oxidative stress contributes to intestinal barrier failure. We studied actions of multispecies probiotics on barrier dysfunction and oxidative stress in experimental AP.Methodology/Principal FindingsFifty-three male Spraque-Dawley rats were randomly allocated into five groups: 1) controls, non-operated, 2) sham-operated, 3) AP, 4) AP and probiotics and 5) AP and placebo. AP was induced by intraductal glycodeoxycholate infusion and intravenous cerulein (6 h). Daily probiotics or placebo were administered intragastrically, starting five days prior to AP. After cerulein infusion, ileal mucosa was collected for measurements of E. coli K12 and 51Cr-EDTA passage in Ussing chambers. Tight junction proteins were investigated by confocal immunofluorescence imaging. Ileal mucosal apoptosis, lipid peroxidation, and glutathione levels were determined and glutamate-cysteine-ligase activity and expression were quantified. AP-induced barrier dysfunction was characterized by epithelial cell apoptosis and alterations of tight junction proteins (i.e. disruption of occludin and claudin-1 and up-regulation of claudin-2) and correlated with lipid peroxidation (r>0.8). Probiotic pre-treatment diminished the AP-induced increase in E. coli passage (probiotics 57.4±33.5 vs. placebo 223.7±93.7 a.u.; P<0.001), 51Cr-EDTA flux (16.7±10.1 vs. 32.1±10.0 cm/s10−6; P<0.005), apoptosis, lipid peroxidation (0.42±0.13 vs. 1.62±0.53 pmol MDA/mg protein; P<0.001), and prevented tight junction protein disruption. AP-induced decline in glutathione was not only prevented (14.33±1.47 vs. 8.82±1.30 nmol/mg protein, P<0.001), but probiotics even increased mucosal glutathione compared with sham rats (14.33±1.47 vs. 10.70±1.74 nmol/mg protein, P<0.001). Glutamate-cysteine-ligase activity, which is rate-limiting in glutathione biosynthesis, was enhanced in probiotic pre-treated animals (probiotics 2.88±1.21 vs. placebo 1.94±0.55 nmol/min/mg protein; P<0.05) coinciding with an increase in mRNA expression of glutamate-cysteine-ligase catalytic (GCLc) and modifier (GCLm) subunits.ConclusionsProbiotic pre-treatment diminished AP-induced intestinal barrier dysfunction and prevented oxidative stress via mechanisms mainly involving mucosal glutathione biosynthesis.

Effect of early antibiotic prophylaxis with ertapenem and meropenem in experimental acute pancreatitis in rats

The clinical course in acute necrotizing pancreatitis is mainly influenced by bacterial infection of pancreatic and peripancreatic necrosis. The effect of two antibiotic treatments for early prophylaxis was studied in the taurocholate model of necrotizing pancreatitis in the rat. Sixty male Sprague-Dawley rats were divided into three pancreatitis groups (15 animals each) and a sham-operated group (15 animals, control group). Pancreatitis was induced by intraductal infusion of 3% taurocholate under sterile conditions. Animals were placed on one of two different antibiotic regimens (15 mg/kg ertapenem or 20 mg/kg meropenem, one shot) after the induction of pancreatitis or received no antibiotics (control). All animals were sacrificed after 24 h to study pancreatic and extrapancreatic infection. Early antibiotic prophylaxis with either erapenam or meropenem significantly decreased pancreatic infection from 12/15 (control group) to 4/15 (ertapenem antibiotic group) and 3/15 (meropenem antibiotic group) (P < 0.05). In our animal model of necrotizing pancreatitis, early antibiotic prophylaxis with ertapenem and meropenem reduced bacterial infection of the pancreas. The efficacy of early antibiotic prophylaxis with ertapenem in the clinical setting should be subject to further research.

Effect of Platelet-Activating Factor Antagonist WEB 2086 on Microcirculatory Disorders in Acute Experimental Pancreatitis of Graded Severity

Platelet-activating factor (PAF) is an important mediator of inflammation and postulated to be involved in the pathogenesis of acute pancreatitis. In this study, we evaluated the therapeutic effect of PAF antagonist WEB 2086 in acute experimental pancreatitis of graded severity in rats. According to a block design, 64 animals were randomly allocated to 8 groups. Severe necrotizing pancreatitis was induced by intraductal infusion of taurocholic acid (4%, 0.4 mL), and the combination of glycodeoxycholic acid (10 mmol/L, 1.0 mL/kg, intraductal infusion) and cerulein (5 microg/kg per hour, intravenous) was applied to induce intermediate pancreatitis, or cerulein alone (5 microg/kg per hour, intravenous) to establish edematous pancreatitis. WEB 2086 was given 15 minutes after beginning the induction of pancreatitis. Pancreatic microcirculation was analyzed in vivo with an epiluminescent microscope. Histopathology was evaluated by a validated score. Trypsinogen-activating peptide and serum amylase were analyzed sequentially. WEB 2086 had no significant influence on the breakdown of microcirculation, leukocyte adherence, histopathological damage, and amylase levels in severe necrotizing pancreatitis, intermediate pancreatitis, and edematous pancreatitis. Only in intermediate pancreatitis was there a significant reduction of trypsinogen-activating peptide levels. In our study, PAF antagonist WEB 2086 had no beneficial effect on microcirculation in acute experimental pancreatitis.

COMBINATION OF ALLOPURINOL AND HYPERBARIC OXYGEN THERAPY

AIM: To investigate the individual and combined effects of allopurinol and hyperbaric oxygen (HBO) therapy on biochemical and histopathological changes, oxidative stress, and bacterial translocation (BT) in the experimental rat acute pancreatitis (AP). METHODS: Eighty-five Sprague-Dawley rats were included in the study. Fifteen of the eighty-five rats were used as controls (sham, GroupI). AP was induced via intraductal taurocholate infusion in the remaining seventy rats. Rats that survived to induction of acute necrotizing pancreatitis were randomized into four groups. Group II received saline, Group III allopurinol, Group IV allopurinol plus HBO and Group V HBO alone. Serum amylase levels, oxidative stress parameters, BT and histopathologic scores were determined. RESULTS: Serum amylase levels were lower in Groups III, IV and V compared to Group II (974 ± 110, 384 ± 40, 851 ± 56, and 1664 ± 234 U/L, respectively, P < 0.05, for all). Combining the two treatment options revealed significantly lower median [25-75 percentiles] histopathological scores when compared to individual administrations (13 [12.5-15] in allopurinol group, 9.5 [7-11.75] in HBO group, and 6 [4.5-7.5] in combined group, P < 0.01). Oxidative stress markers were significantly better in all treatment groups compared to the controls. Bacterial translocation into the pancreas and mesenteric lymph nodes was lower in Groups III, IV and V compared to Group II (54%, 23%, 50% vs 100% for translocation to pancreas, and 62%, 46%, 58% vs 100% for translocation to mesenteric lymph nodes, respectively, P < 0.05 for all). CONCLUSION: The present study confirms the benefit of HBO and allopurinol treatment when administered separately in experimental rat AP. Combination of these treatment options appears to prevent progression of pancreatic injury parameters more effectively.

Probiotics enhance pancreatic glutathione biosynthesis and reduce oxidative stress in experimental acute pancreatitis

Factors determining severity of acute pancreatitis (AP) are poorly understood. Oxidative stress causes acinar cell injury and contributes to the severity, whereas prophylactic probiotics ameliorate experimental pancreatitis. Our objective was to study how probiotics affect oxidative stress, inflammation, and acinar cell injury during the early phase of AP. Fifty-three male Sprague-Dawley rats were randomly allocated into groups: 1) control, 2) sham procedure, 3) AP with no treatment, 4) AP with probiotics, and 5) AP with placebo. AP was induced under general anesthesia by intraductal glycodeoxycholate infusion (15 mM) and intravenous cerulein (5 microg.kg(-1).h(-1), for 6 h). Daily probiotics or placebo were administered intragastrically, starting 5 days prior to AP. After cerulein infusion, pancreas samples were collected for analysis including lipid peroxidation, glutathione, glutamate-cysteine-ligase activity, histological grading of pancreatic injury, and NF-kappaB activation. The severity of pancreatic injury correlated to oxidative damage (r = 0.9) and was ameliorated by probiotics (1.5 vs. placebo 5.5; P = 0.014). AP-induced NF-kappaB activation was reduced by probiotics (0.20 vs. placebo 0.53 OD(450nm)/mg nuclear protein; P < 0.001). Probiotics attenuated AP-induced lipid peroxidation (0.25 vs. placebo 0.51 pmol malondialdehyde/mg protein; P < 0.001). Not only was AP-induced glutathione depletion prevented (8.81 vs. placebo 4.1 micromol/mg protein, P < 0.001), probiotic pretreatment even increased glutathione compared with sham rats (8.81 vs. sham 6.18 miccromol/mg protein, P < 0.001). Biosynthesis of glutathione (glutamate-cysteine-ligase activity) was enhanced in probiotic-pretreated animals. Probiotics enhanced the biosynthesis of glutathione, which may have reduced activation of inflammation and acinar cell injury and ameliorated experimental AP, via a reduction in oxidative stress.

Comparison of the Portal Vein and Kidney Subcapsule as Sites for Primate Islet Autotransplantation

To date, the portal vein has been the primary site for clinical islet transplantation. Despite success, potential complications such as portal vein thrombosis still exist. The kidney subcapsule has been used successfully in rodent models of islet transplantation. We hypothesized that the kidney subcapsule as a site for islet transplantation in the nonhuman primate model would be as effective as the portal vein. Diabetes was induced in the primate Macaca fascicularis via a total pancreatectomy. Animals were kept under anesthesia during the isolation procedure. Islet isolation was performed using intraductal infusion with Liberase HI and mechanical digestion in the Ricordi chamber, and were purified using a continuous Ficoll gradient. Purified islets were autotransplanted either into the portal vein (n = 6) or the left kidney subcapsule (n = 5) of pancreatectomized animals. Intravenous glucose tolerance tests were performed prior to pancreatectomy and 10 days following transplantation. Three animals underwent pancreatectomy and served as diabetic controls. Of the six animals receiving islets in the portal vein, one developed portal vein thrombosis. All remaining autotransplanted animals in this group remained normoglycemic with glucose-induced insulin secretion that was not different from that prior to pancreatectomy. Of the five animals undergoing transplantation into the kidney subcapsule, only one maintained normoglycemia and elicited insulin secretion in response to glucose stimulation. The other four animals remained hyperglycemic. We conclude that the portal vein is superior to the kidney subcapsule as a site for islet transplantation in nonhuman primates 10 days posttransplantation.

Protease-activated Receptor-2 Exerts Contrasting Model-specific Effects on Acute Experimental Pancreatitis

Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.

A polyamine analog bismethylspermine ameliorates severe pancreatitis induced by intraductal infusion of taurodeoxycholate

Stable polyamine homeostasis is important for cell survival and regeneration. Our experimental studies have shown that catabolism of spermidine and spermine to putrescine is associated with the development of pancreatitis. We investigated the pathogenetic role of polyamine catabolism by studying the effect of a methylated polyamine analog on taurodeoxycholate-induced acute experimental pancreatitis. Acute pancreatitis was induced by infusion of sodium taurodeoxycholate (2%) into the pancreatic duct. Bismethylspermine (Me(2)Spm) was administered as a pretreatment before the induction of pancreatitis or as a treatment after the induction of pancreatitis. The sham operation included laparotomy only. Pancreas tissue and blood were sampled at 24 h and 72 h after the infusion of taurodeoxycholate and studied for pancreatitis severity (serum amylase activity, pancreatic water content, and histology) and polyamine catabolism, which includes spermidine/spermine N(1)-acetyltransferase (SSAT) activity as well as spermidine, spermine, and putrescine concentrations in the pancreas. Sodium taurodeoxycholate-induced acute pancreatitis manifests as increases in serum amylase and pancreatic water content, leukocytosis, and acinar cell necrosis in the pancreas. The activity of SSAT increased significantly together with an increase in the ratios of pancreatic putrescine/spermidine and putrescine/spermine at 24 h, which indicates SSAT-induced polyamine catabolism. Pancreatic water content and necrosis were reduced significantly by the treatment with Me(2)Spm at 24 h but not at 72 h when the polyamine homeostasis had recovered, and the pancreatitis had progressed. Taurodeoxycholate-induced acute pancreatitis was associated with activation of polyamine catabolism in the pancreas. The polyamine analog Me(2)Spm ameliorated the injury in the early stage, but it did not ameliorate the late progression of the pancreatic necrosis at 72 h. Thus, besides proteolytic enzyme activation and the cascades of inflammation, polyamine catabolism may be an important pathogenetic mediator of the early stages of acute pancreatitis.