Characterization of ischemia/reperfusion injury after pancreas transplantation and reduction by application of monoclonal antibodies against ICAM-1 in the rat

BACKGROUND: Reperfusion injury contributes to early organ malfunction after transplantation. The aim of this study was to characterize the ischemia-reperfusion injury after pancreas transplantation and to evaluate the effect of monoclonal antibody therapy against ICAM-1. METHODS: Twenty-five heterotopic pancreas transplantations were performed in syngenic rats in a no-touch technique. Microcirculation and leukocyte-endothelial interaction in the grafts were evaluated by intravital microscopy at 1 hour (I), 6 hours (II), 12 hours (III), and 24 hours (IV) after reperfusion, and 12 hours after reperfusion (V) in the therapeutic group. In (V) antibodies against ICAM-1 were applied as bolus at reperfusion and continuously for 6 hours. Next to intravital microscopy, histologic scores, myeloperoxidase levels, and ICAM-1 expression were determined. RESULTS: Microcirculation was significantly reduced in capillaries and postcapillary venules in the time course after reperfusion (capillary: 0.96 +/- 0.08 mm/s [I] to 0.45 +/- 0.07 mm/s [IV] [P <.01]) and was improved significantly by therapy with ICAM-1 antibodies (0.98 +/- 0.06 mm/s, (P <.01). Leukocyte-endothelial interaction significantly increased 6 hours after reperfusion (II) (P <.01). These changes were paralleled by an increased endothelial expression of ICAM-1 in immunohistochemistry. Histologic evaluation showed increased inflammation at 12 hours after reperfusion (P <.01), which could be diminished by the administration of ICAM-1 antibodies (P <.05). CONCLUSION: Increased endothelial expression of ICAM-1 after pancreas transplantation is positively correlated with microcirculatory impairment. Important steps of pancreatic inflammation take place approximately 6 hours after reperfusion. Prophylactic application of monoclonal antibodies against ICAM-1 reduces reperfusion injury and successfully prevents the occurrence of graft pancreatitis.     

Characterization and reduction of ischemia/reperfusion injury after experimental pancreas transplantation

Reperfusion injury after pancreas transplantation is a cause of early graft pancreatitis. The aim of this study was to quantify pancreatic microcirculation after pancreas transplantation in correlation with cold ischemia time. In a second step the effect of N-acetylcysteine on reperfusion damage was tested. Pancreas transplantation was performed in three different groups of male Lewis rats. Groups 1 and 2 received no special treatment. Cold ischemia time was 1.5 hours in group 1 and 16 hours in groups 2 and 3. In group 3 donor and recipient were both treated with N-acetylcysteine (300 mg/kg) 1.5 hours after reperfusion graft microcirculation was quantified by means of intravital microscopy. Rhodamine-labeled leukocytes, fluoroscein isothiocyanate-labeled erythrocytes, and fluoroscein isothiocyanate-albumin were used as fluorochromes. After a cold ischemia time of 16 hours, functional capillary density, erythrocyte velocity, and leukocyte-endothelium interaction were reduced significantly compared to a cold ischemia time of 1.5 hours (P <0.05). After 16 hours of cold ischemia, treatment with N-acetylcysteine improved all of these parameters (P ≤ 0.05). Ischemia/reperfusion injury after experimental pancreas transplantation is characterized by a disturbance of the pancreatic microcirculation exhibiting a correlation with the duration of cold ischemia. Treatment of donor and recipient with N-acetylcysteine resulted in prevention of cold ischemia-induced microcirculatory disturbance. Supported by "Forschungsschwerpunkt Transplantation," Baden-Württemberg, Germany. Presented at the Thirty-Ninth Annual Meeting of The Society for Surgery of the Alimentary Tract, New Orleans, La., May 17–20, 1998.     

Effects of heparin in experimental models of acute pancreatitis and post-ERCP pancreatitis

BACKGROUND: Acute pancreatitis (AP) is a complication of diagnostic or therapeutic endoscopic retrograde cholangiopancreatography (ERCP). In a recent clinical trial, a decreased rate of post-ERCP pancreatitis was shown after prophylactic heparin treatment. The aim of this study was to evaluate the effects of prophylactic heparin application in various experimental models of AP and pancreatic duct obstruction and to assess the underlying mechanisms. METHODS: In various experimental models, pancreatic injury of graded severity was induced in Wistar rats: (1) mild pancreatitis by IV cerulein infusion over 6 hours; (2) severe pancreatitis by infusion of glycodeoxycholic acid into the pancreatic duct plus IV cerulein application over 6 hours. The clinical ERCP situation was imitated in groups (3) obstruction of the pancreatic duct and (4) infusion of contrast medium into the pancreatic duct plus obstruction. In every group the animals received either no heparin (n=six per group) or continuous IV heparin (n=six per group) starting before pancreatic injury. Histologic changes, amylase, and lipase in plasma were evaluated 12 hours after induction of pancreatic injury. Additional animals were treated to investigate pancreatic microcirculation by intravital microscopy (n=six per group). RESULTS: In groups 1, 3, and 4 (mild AP/duct obstruction/duct obstruction plus contrast medium), IV heparin-treated animals showed reduced edema, inflammation, and peak amylase values compared with the corresponding non-heparin-treated animals (P<.05). Moreover, mean erythrocyte velocity was significantly higher and leukocyte-endothelium interaction was reduced in these groups after prophylactic administration of heparin. In contrast, group 2 (severe AP) did not show any difference between control animals and animals that received heparin as assessed by histology and intravital microscopy. CONCLUSIONS: Prophylactic systemic application of heparin provides a protective effect in mild AP and in experimental post-ERCP pancreatitis. The mechanism of the protective effects of heparin seems to be the reduction of leukocyte-endothelium interaction and the normalization of pancreatic microcirculation.     

Donor Pretreatment with Tetrahydrobiopterin Saves Pancreatic Isografts from Ischemia Reperfusion Injury in a Mouse Model

Depletion of the nitric oxide synthase cofactor tetrahydrobiopterin (H4B) during ischemia and reperfusion is associated with severe graft pancreatitis. Since clinically feasible approaches to prevent ischemia reperfusion injury (IRI) by H4B‐substitution are missing we investigated its therapeutic potential in a murine pancreas transplantation model using different treatment regimens. Grafts were subjected to 16 h cold ischemia time (CIT) and different treatment regimens: no treatment, 160 μM H4B to perfusion solution, H4B 50 mg/kg prior to reperfusion and H4B 50 mg/kg before recovery of organs. Nontransplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2 h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by high pressure liquid chromatography (HPLC). Compared to nontransplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pretreatment with H4B resulted in almost completely abrogated IRI‐related damage showing graft microcirculation comparable to nontransplanted controls and restored intragraft H4B levels, resulting in significant reduction of parenchymal damage (p < 0.002) and improved survival and endocrine function (p = 0.0002 each). H4B donor pretreatment abrogates ischemia‐induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation.     

Experimental studies on the effects of superoxide dismutase on warm ischemic-reperfusion injury of the lung]

Transient impairment of the transplanted lung in early postoperative period is one of difficult problems in lung transplantation. It is likely that reperfusion injury of the warm ischemic lung is contributory. The purpose of this study is to evaluate the effects of superoxide dismutase (SOD) on reperfusion injury of warm ischemic lung. Thirty mongrel dogs were divided into four groups. In group I (n = 6), the left lung with complete hilar stripping was placed in warm ischemic state under deflation for 1 hour. In group II (n = 9), the left lung with complete hilar stripping was kept in warm ischemic condition under inflation. Group III (n = 6) animals with same manipulation as group I received superoxide dismutase (SOD 20 mg/kg) before reperfusion. Group IV (n = 9) animals underwent same manipulation as group II and received SOD (20 mg/kg) before reperfusion. Before warm ischemia, immediately after reperfusion, and 1 and 2 hours, blood gases, left pulmonary vascular resistance were measured under the occlusion of right pulmonary artery. Extra vascular lung water content (EVLW) was measured at autopsy and lung was processed for histology. In group II, III and IV, blood gases and EVLW showed significantly better values than group I. In group I and III, left pulmonary vascular resistance increased prominently after reperfusion, however did not change in group II and IV. From these results, we concluded that inflated lung reduced the extent of pulmonary edema after reperfusion and SOD was effective in preventing warm ischemic damage even in deflated lung.     

Attenuation of microvascular reperfusion injury in rat pancreas transplantation by L-arginine

BACKGROUND: Despite improving results, exocrine complications remain a major challenge in clinical pancreas transplantation. The etiology of posttransplantation pancreatitis relates almost certainly to cold ischemia/reperfusion-induced microvascular injury with an imbalance of vasoconstricting and vasodilating mediators due to endothelial dysfunction. We therefore studied the effectiveness of a nitric oxide donor on postischemic microvascular reperfusion injury after pancreas transplantation. METHODS: Heterotopic isogeneic pancreaticoduodenal transplantation was performed in spontaneously breathing, chloralhydrate-anesthetized Sprague Dawley rats after 16 hr (n=5) of cold storage of the graft in 4 degrees C histidine-tryptophane-ketoglutarate solution. An additional five animals received L-arginine immediately before (50 mg/kg i.v.) and during the first 30 min of reperfusion (100 mg/kg i.v.). Five animals that did not undergo transplantation served as controls. Intravital fluorescence microscopy was used for analysis of functional capillary density, capillary diameters, and capillary red blood cell velocity in exocrine pancreatic tissue during 120 min of reperfusion. Histology served for quantitative assessment of inflammatory response (leukocytic tissue infiltration) and endothelial disintegration (edema formation). RESULTS: In L-arginine-treated animals, functional capillary density of exocrine tissue of pancreatic grafts was found slightly higher after 30 and 60 min, and significantly higher after 120 min of postischemic reperfusion compared with untreated pancreatic grafts. This was accompanied by a significant increase of capillary diameters. In parallel, pancreatic histology revealed significant attenuation of both leukocytic tissue infiltration and edema formation in the L-arginine-treated animals when compared with the nontreated controls. CONCLUSIONS: Besides reduction of leukocyte-dependent microvascular injury, L-arginine improves postischemic microvascular reperfusion, supposedly by capillary dilatation. Thus, our results suggest that supplement of nitric oxide during reperfusion is effective in attenuating exocrine microvascular reperfusion injury.     

Ischemic preconditioning fails to improve microcirculation but increases apoptotic cell death in experimental pancreas transplantation

Brief periods of warm ischemia and subsequent short reperfusion before either long-term cold or warm ischemic insult (ischemic preconditioning, IPC) have proven to ameliorate ischemia/reperfusion (I/R) injury in various organs, such as the liver and lung. The aim of this study was to examine the effect of IPC on pancreatic cell apoptosis and microcirculatory impairments in experimental pancreas transplantation. Male Lewis rats served as donors and recipients of heterotopic syngeneic pancreaticoduodenal transplantation. Recipient animals were divided into two experimental groups: group Tx (n=7) received grafts without IPC, group Tx&IPC received grafts with IPC. Animals that had not undergone transplantation but whose pancreata had been exteriorized served as controls (n=5). All pancreatic grafts were preserved in University of Wisconsin solution for 6 h at 4°C. IPC was induced by interruption of the arterial blood flow for 10 min followed by 10 min of reperfusion. One and two hours after reperfusion, graft microcirculation was assessed by means of intravital microscopy (IVM). Rats were immediately killed after the second measurement and DNA breaks of acinar cells were detected by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end-labelling (TUNEL) assay and gel electrophoresis (laddering). The apoptotic index (AI) was defined as the number of apoptotic cells per high-power field. Analysis of both groups of transplanted grafts showed a significant decrease in functional capillary density (FCD) and a significant increase in leukocyte sticking to postcapillary venules (LAV) at 1 h and 2 h of reperfusion, compared with animals that had not undergone transplantation (P<0.01). In parallel, AI was significantly increased in transplanted grafts compared to the controls (P<0.01). Grafts subjected to IPC showed no significant differences, neither for FCD nor LAV, at both time points if compared with grafts of group Tx. However, IPC resulted in a significant increase in AI (P<0.05). We can conclude that IPC has no effect on pancreatic microcirculation but enhances acinar cell apoptosis in experimental pancreas transplantation. These results indicate that IPC might increase I/R injury after pancreatic cold ischemia.     

Organ procurement in experimental pancreas transplantation with minimal microcirculatory impairment

BACKGROUND: Ischemia-reperfusion injury has been shown to deteriorate microcirculation in experimental pancreas transplantation. However, minor concern was taken on the impact of organ procurement in this condition. We examined the impact of a standardized technique of organ procurement on microcirculation and apoptosis in experimental pancreas transplantation. METHODS: Male Lewis rats were divided into three groups: sham-operated animals without dissection of the pancreas served as controls (n = 5); animals undergoing nearly total process of organ procurement with the pancreas pedunculated on the aorta and the hepatoduodenal ligament (n = 7), and animals receiving pancreaticoduodenal transplantation. Pancreatic grafts were preserved for 6 h in cold University of Wisconsin solution (n = 7). At 1 and 2 h reperfusion and in time-matched controls, microcirculation was assessed by means of intravital fluorescence microscopy. Tissue samples were obtained after 2 h measurement and DNA breaks of acinar cells were detected by in situ nick end-labeling (TUNEL assay). The apoptotic index (apoptotic cells per high- power fields; hpf) was quantified by microscopic counting of at least 50 hpf. RESULTS: Assessment of functional capillary density (FCD) in animals undergoing subtotal process of organ procurement revealed a slight non-significant decrease at 1 and 2 h compared with controls. In addition, leukocyte sticking to postcapillary venules (LAV) as well as the apoptotic index were found slightly increased after organ procurement compared with controls (p > 0.05). However, after pancreas transplantation the apoptotic index and the LAV were significantly increased and the FCD significantly decreased compared with both groups of non-transplanted animals (p < 0.01). CONCLUSIONS: Our validated technique of organ procurement does not negatively impact microcirculation and apoptosis in experimental pancreas transplantation. Copyright 2004 S. Karger AG, Basel     

Electron microscopy of the exocrine pancreas in experimental acute hypercalcemia

Hypercalcemia has been associated with acute pancreatitis clinically and in the experimental animal. We studied the pancreatic ultrastructure in acute experimental hypercalcemia. Anesthetized cats (Pentobarbital, 0.55 mg/kg) received Ca++ (Calcium-Gluconate: 0.6 mmol/kgh; n = 4), K+ (KCl: 1.1 mmol/kgh; n = 4) or NaCl (0.9%; n = 4) locally through retrograde infusion into the splenic artery. Biopsies for electron microscopy (EM) were taken at three hours. Eight cats received intravenous Ca++ (0.6 mmol/kgh, 0.3 mmol/kgh after three hours) or NaCl (0.9%) for 12 hours. Biopsies were collected in two animals in three-hour intervals, and in all animals at twelve hours. After local calcium infusion necrotizing pancreatitis was seen macroscopically in the body of the pancreas. Biopsies for EM showed acinar cell necrosis, hydrops of nuclei and mitochondria and needle-like precipitates in the cytoplasma in the center of calcium perfusion. Biopsies taken from the peripheral region of the macroscopically altered tissue revealed desorganisation of the acinar polarisation and the endoplasmic reticulum, with zymogen granules appearing in the basolateral cell-portion. After intravenous calcium administration no macroscopical changes were seen. In EM acinar cells showed dilatation and proliferation of the golgi apparatus and increased number of condensing vacuoles indicating stimulation. Again, disorganisation of acinar cell polarisation was present. Control animals treated with K+ or NaCl showed normal pancreatic ultrastructure. The morphological changes after calcium infusion indicate direct damage to the acinar cell. Our results suggest that hypercalcemia induced pancreatitis could originate in the acinar cell.     

Targeting vascular endothelial growth factor pathway offers new possibilities to counteract microvascular disturbances during ischemia/reperfusion of the pancreas

BACKGROUND: It is of crucial importance to explore new therapeutic strategies capable of combating or even preventing pancreatic graft failure after transplantation caused by ischemia reperfusion damage. So far, the role of the hypoxia induced mediator vascular endothelial growth factor (VEGF) upon pancreatic microcirculation has not been described. Therefore the aim of this study was to investigate its influence, using the novel tyrosinekinase inhibitor PTK787/ZK222584 (PTK/ZK), upon functional capillary density (FCD), leukocyte-endothelium interaction (LEI), and macromolecular permeability (P) of normal and postischemic pancreas tissue. METHODS: Sprague-Dawley rats were anesthetized and randomly assigned to five groups (n=7/group): (a) sham, (b) ischemia/reperfusion (I/R) control, (c) I/R and PTK/ZK treatment, (d) VEGF-superfusion, (e) VEGF-superfusion and PTK/ZK-treatment. A recently established method of digital dynamic intravital epifluorescence microscopy was used for evaluating the effective microvascular permeability together with FCD and LEI. RESULTS: Comparison between sham vs. I/R shows a significant upregulation of VEGF-expression followed by deterioration of microcirculation with decreased FCD, increased P and LEI. Treatment with PTK/ZK resulted in a significant decrease of P under conditions of superfusion with VEGF as well as I/R compared to corresponding groups without treatment. CONCLUSION: VEGF plays a crucial causative role involving an increase in permeability in normal as well as in postischemic pancreatitis via tyrosinkinase receptors. VEGF seems to be partly accountable for a deterioration of FCD and an upregulation of LEI via VEGF-tyrosinekinase receptor independent mechanisms. VEGF might be a promising potential therapeutic target in order to minimize edema formation caused by I/R and pancreatitis in pancreas transplantation.     

Antithrombin therapy in pancreas retransplantation and pancreas-after-kidney/pancreas-transplantation-alone patients

Fertmann JM, Arbogast HP, Illner W‐D, Tarabichi A, Dieterle C, Land W, Jauch K‐W, Hoffmann JN. Antithrombin therapy in pancreas retransplantation and pancreas‐after‐kidney/pancreas‐transplantation‐alone patients.
Clin Transplant 2011: 25: E499–E508. © 2011 John Wiley & Sons A/S. Abstract: Antithrombin (AT) is a coagulatory inhibitor with pleiotropic activities. AT reduces ischemia/reperfusion injury and has been successfully used in patients with simultaneous pancreas kidney transplantation. This study retrospectively analyzes prophylactic high‐dose AT application in patients with solitary pancreas transplantation traditionally related to suboptimal results. In our center, 31 patients received solitary pancreas transplantation between 7/1994 and 7/2005 (pancreas retransplantation, PAK/PTA). The perioperative treatment protocol was modified in 5/2002 now including application of 3000 IU. AT was given intravenously before pancreatic reperfusion (AT, n = 18). Patients receiving standard therapy served as controls (n = 13). Daily blood sampling was performed during five postoperative days. Standard coagulatory parameters and number of transfused red blood cell units were not altered by AT. In AT patients serum amylase (p < 0.01) and lipase (p < 0.01) on postoperative days 1, 2 and 3 were significantly reduced. Our actual perioperative management protocol including high dose AT application in human solitary pancreas transplantation reduced postoperative liberation of pancreatic enzymes in this pilot study. Prophylactic AT application should deserve further clinical testing in a randomized controlled trial.     

Adenosine analogue reduces spinal cord reperfusion injury in a time-dependent fashion

BACKGROUND: We hypothesized that inflammation during spinal cord reperfusion worsens ischemic injury. ATL-146e, an adenosine A(2A) agonist with known anti-inflammatory properties, was used to test this hypothesis at varied intervals to determine the time course of reperfusion injury. METHODS: Forty rabbits underwent cross-clamping of the infrarenal aorta for 45 minutes. One group (n = 14 animals) received 0.06 microg/kg/min systemic ATL-146e over 3 hours, beginning after 30 minutes of ischemic time. A second group (n = 6 animals) received ATL-146e over 1.5 hours. A third group (n = 3 animals) received ATL-146e over 1 hour, and a fourth group (n = 17 animals) received saline solution. All animals were assessed at 48 hours for hind limb motor function (Tarlov scale, 0-5). RESULTS: Animals that received ATL-146e for 3 hours (Tarlov score, 4.3 +/- 0.22; P <.001) or 1.5 hours (Tarlov score, 2.7 +/- 0.6; P <.05) had improved neurologic outcomes compared with rabbits that received saline solution (Tarlov score, 0.6 +/- 0.29). Animals that received ATL-146e for 1 hour (Tarlov score, 0.7 +/- 0.8) were not significantly different from those animals that received saline solution. CONCLUSIONS: Systemic ATL-146e, given during reperfusion, results in time-dependent improvement in spinal cord function after ischemia. This implies that the mechanism of spinal reperfusion injury includes leukocyte-mediated inflammation at a critical post-ischemic time interval.     

Effects of Organ Preservation, Ischemia Time and Caspase Inhibition on Apoptosis and Microcirculation in Rat Pancreas Transplantation

This study was undertaken to examine the impact of ischemia-reperfusion (I/R) injury on microcirculation and apoptosis in experimental pancreas transplantation. Pancreatic grafts were subjected to different preservation solutions and cold ischemia times (CITs): University of Wisconsin (UW), 6-h CITs (group U6); UW, 18-h CITs (group U18); normal saline, 6-h CITs (group S6); and normal saline, 6-h CITs with Z-Asp-2,6-dichlorobenzoyloxymethylketone (pan-caspase inhibitor; group S6 & CI). Nontransplanted animals served as controls. At 1- and 2-h reperfusion microcirculation was assessed by means of intravital microscopy. Apoptosis was detected by in situ nick end-labeling method (TUNEL) at 2-h reperfusion. Deterioration of microcirculation was lowest in group U6 and highest in groups S6 and S6 & CI compared with controls. The apoptotic index (cells per high power fields) of groups U6, U18 and S6 correlated well with functional capillary density (r=- 0,70, p < 0.0001) and leucocyte sticking (r= 0,69, p < 0.0001) at 1-h reperfusion. Caspase inhibition had no impact on microcirculation but significantly reduced AI compared with group S6 (p < 0.001). These data suggest that pancreatic I/R injury-induced apoptotic cell death well predicts the extent of [corrected] microcirculatory impairment. Caspase inhibition might be a promising strategy in reducing I/R injury in pancreas transplantation.     

Systemic intravenous infusion of bovine hemoglobin significantly reduces microcirculatory dysfunction in experimentally induced pancreatitis in the rat

OBJECTIVE: To evaluate the effectiveness of bovine hemoglobin on pancreatic microcirculation and outcome in experimental acute rodent pancreatitis. SUMMARY BACKGROUND DATA: Stasis of the pancreatic microcirculation initiates and aggravates acute pancreatitis. Hydroxyethyl-starch (HES) has been shown to improve pancreatic microcirculation. Similarly, bovine hemoglobin might improve rheology due to its colloid effect, but additionally supplies oxygen to oxygen depleted pancreatic tissue. METHODS: In Wistar rats, severe acute pancreatitis was induced by administration of glucodeoxycholic acid i.d. and cerulein i.v. Pancreatic microcirculation was continuously monitored by fluorescence microscopy. Fifteen minutes after the initiation of acute pancreatitis, animals received either 0.8 mL bovine hemoglobin (Oxyglobin), HES, or 2.4 mL 0.9% NaCl i.v. at random. After 6 hours, animals were killed and histopathological damage of the pancreas was assessed using a validated histology score (0-16). RESULTS: In comparison to controls, pancreatic microcirculation improved significantly in the HBOC group (mean difference of capillary density 31.4%; standard error 5.6%; P < 0.001; 95% confidence interval for difference 17.5-45.3). HES was not as effective as HBOC substitution. The histology score revealed less tissue damage in the HBOC group [6.25 vs. 9.25 (3-8.5 vs. 8-10.75, P < 0.001)] in comparison to controls and also in comparison to the HES group [6.25 vs. 8 (3-8.5 vs. 6.5-10.25, P < 0.006)]. CONCLUSIONS: In severe acute pancreatitis, single i.v. injection of bovine hemoglobin improves pancreatic microcirculation and reduces tissue damage.     

Extrapancreatic organ impairment in caerulein induced pancreatitis.

BACKGROUND AND AIMS: Multiorgan function failures are the major fatal complications in acute pancreatitis. In this experiment, we studied 1) the manifestation and time course of extrapancreatic organ damage in an acute pancreatitis model and 2) whether the obstructive liver damage in this model is caused by the obstruction of common biliopancreatic duct compressed by oedematous pancreas. MATERIAL AND METHODS: 80 male Wistar rats were divided into two groups: control and caerulein groups (five subgroups in each group). In the caerulein group, the acute pancreatitis was induced by caerulein intraperitoneal injections. In the controls equal volume of saline was injected. Two subgroups, one in caerulein and one in control groups, had an intrapancreatic bile duct stent inserted transduodenally before the injections. The pancreas, liver, lung and kidney tissues and blood samples were obtained for the measurement or analysis of interstitial oedema, plasma amylase, alanine aminotransferase, bilirubin, urea, creatinine, alkaline phosphatase, lactate dehydrogenase, blood gas and electron microscopy at 1, 6, 12 and 24 hours after the last injection in unstented animals, and at 6 hours in stented animals. RESULTS: Lungs and kidney remained unchanged. Liver damage was found during the first 6-12 hours, manifest as increased plasma alanine aminotransferase and bilirubin and dilatation of bile canaliculi and hepatocyte damage in electron microscopy. The intrapancreatic bile duct stent did not resolve these changes. CONCLUSIONS: The liver may be the first evolved extrapancreatic organ in the early stage in this mild oedematous pancreatitis model and the hepatocyte damage is not caused by the obstruction of common biliopancreatic duct compressed by the oedematous pancreas.     

Whole gut washout ameliorates the progression of acute experimental pancreatitis

BACKGROUND: Septic complications are mainly responsible for deterioration of a patient with acute pancreatitis. Intestinal tract is accepted as the main source of pancreatic or peripancreatic infection. MATERIAL AND METHODS: Acute pancreatitis was induced in 40 Sprague-Dawley rats by ligation of the main biliopancreatic duct. Animals were divided into two groups. The first group of animals (n = 20) received high volume polyethylene glycol-3500 (GoLYTELY) for 6 hours through a silastic catheter introduced into the proximal part of the jejunum from a puncture gastrostomy during the initial laparotomy. The second group animals (n = 20) did not receive any treatment. Half of the animals from each group were sacrificed 72 hours later and tissue samples were taken from mesenteric lymph nodes, pancreas, spleen, and liver for bacteriologic cultures. Cecum cultures were also prepared. Blood samples at 72 hours were obtained for the measurement of amylase, lactic dehydrogenase (LDH), lactic acid, alanine aminotransferase (ALT), glucose, calcium, arterial pH, base excess, partial oxygen pressure, bicarbonate, leucocyte count, and hematocrit levels. The pancreas was examined histopathologically. The remaining half of the animals from each group were allowed to survive until death. RESULTS: The levels of amylase, LDH, ALT, lactic acid, pH, pO(2), bicarbonate and base excess for the rats in group I were significantly lower when compared with the rats in group II (P<0.05). Positive mesenteric lymph node cultures were detected in 30% of group I animals whereas they were positive in 90% of group II animals (P = 0.0198). Distant organ cultures were positive in 8 animals (liver 5, spleen 2, pancreas 1) in group II, whereas only one positive distant organ culture (liver) was established in group I (P>0.05). Histopathological scoring observed in the pancreas were less severe for the rats in group I when compared with the rats in group II (P = 0.012). The rats in group I survived longer than the rats in group II (median survival 6.8 days versus 17.3 days, P<0.001). CONCLUSIONS: Whole gut washout with high-volume polyethylene glycol in pancreatitis reduced the blood levels of enzymes and increased the survival. Whole gut washout for acute pancreatitis appears effective to ameliorate the prognostic factors in blood and this modality may be a promising treatment method in acute pancreatitis.     

C1-esterase inhibitor reduces reperfusion injury after lung transplantation

BACKGROUND: Activation of the complement system and polymorphonuclear neutrophilic leukocytes plays a major role in mediating reperfusion injury after lung transplantation. We hypothesized that early interference with complement activation would reduce lung reperfusion injury after transplantation. METHODS: Unilateral left lung autotransplantation was performed in 6 sheep. After hilar stripping the left lung was flushed with Euro-Collins solution and preserved for 2 hours in situ at 15 degrees C. After reperfusion the right main bronchus and pulmonary artery were occluded, leaving the animal dependent on the reperfused lung (reperfused group). C1-esterase inhibitor group animals (n = 6) received 200 U/kg body weight of C1-esterase inhibitor as a short infusion, half 10 minutes before, the other half 10 minutes after reperfusion. Controls (n = 6) underwent hilar preparation only. Pulmonary function was assessed by alveolar-arterial oxygen difference and pulmonary vascular resistance. The release of beta-N-acetylglucosaminidase served as indicator of polymorphonuclear neutrophilic leukocyte activation. Extravascular lung water was an indicator for pulmonary edema formation. Biopsy specimens were taken from all groups 3 hours after reperfusion for light and electron microscopy. RESULTS: In the reperfused group, alveolar-arterial oxygen difference and pulmonary vascular resistance were significantly elevated after reperfusion. All animals developed frank alveolar edema. The biochemical marker beta-N-acetylglucosaminidase showed significant leukocyte activation. In the C1-esterase inhibitor group, alveolar-arterial oxygen difference, pulmonary vascular resistance, and the level of polymorphonuclear neutrophilic leukocyte activation were significantly lower. CONCLUSIONS: Treatment with C1-esterase inhibitor reduces reperfusion injury and improves pulmonary function in this experimental model.     

Antithrombin III pretreatment reduces neutrophil recruitment into the lung and skeletal muscle tissues in the rat model of bilateral lower limb ischemia and reperfusion: a pilot study

BACKGROUND: Anti-inflammatory action of Antithrombin III (AT III) is still not well understood in ischemia/reperfusion (I/R) injury. In the present study, we aimed to investigate the anti-inflammatory action of AT III on remote lung and local skeletal muscle tissue injury in a rat model of bilateral lower limb I/R model. METHODS: Bilateral lower limb ischemia and reperfusion were produced by means of tourniquets occlusions and releases, respectively. Three groups of rats were used in this controlled study: sham group (sham, n=3) underwent 5 h of anesthesia only; control group (I/R, n=7) underwent 3 h of bilateral lower limb ischemia followed by 2 h of reperfusion; and AT III pretreated group (I/R-AT III, n=6) underwent the same procedure as the control group, but also received i.v. 250 U kg-1 AT III 30 min before ischemia induction under midazolam and fentanyl anesthesia. MEASUREMENTS AND RESULTS: Lung and muscle tissue accumulation of polymorphonuclear leukocytes (PMN) were assessed by measuring tissue myeloperoxidase (MPO) activity. Histopathological changes in tissues were assessed by PMN counts in the lung, and muscle tissues and by histological lung injury score. Plasma 6-keto prostaglandin F(1alpha) and tumor necrosis factor alpha levels were measured by an enzyme immunoassay technique. Myeloperoxidase activity could not be detected in the muscle tissues of all groups. The lung and muscle tissue PMN counts in the I/R group were significantly higher compared with the I/R-AT III group (P<0.05). CONCLUSIONS: Data from the present study provides some evidence that AT III pretreatment attenuates remote lung and local skeletal muscle tissue injury caused by lower limb I/R.