Treatment With Tetrahydrobiopterin Overcomes Brain Death–Associated Injury in a Murine Model of Pancreas Transplantation

Brain death (BD) has been associated with an immunological priming of donor organs and is thought to exacerbate ischemia reperfusion injury (IRI). Recently, we showed that the essential nitric oxide synthase co‐factor tetrahydrobiopterin (BH4) abrogates IRI following experimental pancreas transplantation. We therefore studied the effects of BD in a murine model of syngeneic pancreas transplantation and tested the therapeutic potential of BH4 treatment. Compared with sham‐operated controls, donor BD resulted in intragraft inflammation reflected by induced IL‐1ß, IL‐6, VCAM‐1, and P‐selectin mRNA expression levels and impaired microcirculation after reperfusion (p < 0.05), whereas pretreatment of the BD donor with BH4 significantly improved microcirculation after reperfusion (p < 0.05). Moreover, BD had a devastating impact on cell viability, whereas BH4‐treated grafts showed a significantly higher percentage of viable cells (p < 0.001). Early parenchymal damage in pancreatic grafts was significantly more pronounced in organs from BD donors than from sham or non‐BD donors (p < 0.05), but BH4 pretreatment significantly ameliorated necrotic lesions in BD organs (p < 0.05). Pretreatment of the BD donor with BH4 resulted in significant recipient survival (p < 0.05). Our data provide novel insights into the impact of BD on pancreatic isografts, further demonstrating the potential of donor pretreatment strategies including BH4 for preventing BD‐associated injury after transplantation.     

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.     

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     

Intermittent capillary perfusion in rat pancreas grafts following short- and long-term preservation in University of Wisconsin solution

In pancreas transplantation (PTx), ischemia/reperfusion-induced deterioration of graft-microcirculation is accompanied by alterations of intermittent capillary perfusion (IP; alternating cessation and resumption of capillary blood flow) is known to counteract malperfusion. Incidence and effectiveness of IP following short- versus long-term preservation of pancreas grafts with University of Wisconsin (UW) solution has not been examined so far. PTx was performed in Lewis rats following 2-h or 18-h preservation in UW solution. Using intravital fluorescence microscopy, functional capillary density (FCD), red blood cell (RBC) velocity, IP-incidence and -frequency were analyzed. Laser Doppler flowmetry allowed for the determination of erythrocyte flux and velocity. Measurements were performed at 30, 60 and 120 min after reperfusion. Nontransplanted animals served as controls. FCD, RBC-velocity and -flux remained unchanged in the 2-h group. IP was encountered in 87% of all observation areas at 120 min. After 18-h ischemia, FCD was significantly reduced, which was paralleled by a 50% incidence of IP at 120 min. Tissue edema and leukocyte infiltration in pancreas grafts following 18-h preservation were significantly enhanced. Therefore, IP is an important mechanism aimed at improving microcirculation and UW solution is suitable to preserve vasomotion-activities enabling long-term preservation in a pancreas graft.     

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.     

In Vivo Imaging of Human Pancreatic Microcirculation and Pancreatic Tissue Injury in Clinical Pancreas Transplantation

Pancreatitis remains to be a major complication following clinical pancreas transplantation. We performed orthogonal polarized spectral (OPS) imaging for direct in vivo visualization and quantification of human pancreatic microcirculation in six healthy donors for living donor liver transplantation and 13 patients undergoing simultaneous pancreas-kidney transplantation. We further determined the impact of microvascular dysfunction during early reperfusion on pancreatic graft injury. Exocrine and endocrine pancreatic impairment was determined by analysis of serum lipase, amylase and C-peptide levels. Compared to normal pancreas in liver donors (homogeneous acinar perfusion) functional capillary density (FCD) and capillary red blood flow velocity of reperfused grafts were significantly decreased. Elevated CRP concentrations on day 2 post-transplant and serum lipase and amylase levels determined on days 4-5 significantly correlated with microvascular dysfunction during the first 30 min of graft reperfusion. Post-transplant serum C-peptide also correlated significantly with pancreatic capillary perfusion. OPS imaging allows to intra-operatively assess physiologic pancreatic microcirculation and to determine microcirculatory impairment during early graft reperfusion. This impairment correlated with the manifestation of post-transplant dysfunction of both exocrine and endocrine pancreatic tissue. OPS imaging may be used clinically to determine the efficacy of interventions, aiming at attenuating microcirculatory impairment during the acute post-transplant reperfusion phase.     

Exocrine, but not endocrine, tissue is susceptible to microvascular ischemia/reperfusion injury following pancreas transplantation in the rat

While post-transplant pancreatitis is still a frequently occurring complication of whole pancreas transplantation, dysfunction of the endocrine tissue is rarely observed. Given that microcirculatory disorders play a major role in the pathogenesis of pancreatitis, we hypothesized a dissociation of endocrine and exocrine microvascular control in pancreas transplantation (cold ischemia-reperfusion) and studied this dissociation quantitatively, analyzing the pancreatic microcirculation after heterotopic isogeneic pancreaticoduodenal transplantation in rats by means of fluorescence microscopy. Functional capillary density (FCD) of both exocrine and endocrine tissue of pancreatic grafts after 1 h of cold storage in HTK solution did not differ when compared to sham-operated, time-matched controls. Intermittent capillary perfusion, which is absent under sham control conditions and which is proposed to be operative as a compensatory mechanism to counteract malperfusion, was observed in 52 % of the exocrine, but in only 8 % of the endocrine, tissue studied (p < 0.05). In contrast, cold storage of pancreatic grafts for 6 h in HTK resulted in a complete loss of intermittent capillary perfusion in exocrine tissue and, consequently, marked exocrine perfusion failure (decrease in FCD), while FCD of pancreatic endocrine tissue was preserved without any significant change in the incidence of intermittent capillary perfusion. Thus, our results indicate a higher susceptibility of the exocrine pancreas to cold ischemia/reperfusion events that is associated with significant alterations in nutritive perfusion and, thus, with limitations of the oxygen supply to the tissue. This may lead to inflammatory tissue reactions in the clinical setting of pancreas transplantation. Received: 4 June 1998 Received after revision: 17 September 1998 Accepted: 25 September 1998     

Brain Death Impairs Pancreatic Microcirculation

Brain death (BD) influences the quality of donor grafts in transplantation. To evaluate the impact of BD on pancreas grafts, we investigated the influence of BD on the microcirculation and histology of the pancreas in a rat model of explosive BD. A group of Wistar rats (n=7), rendered brain dead by inflating an intracranially inserted Fogarty catheter was compared with controls (CO) using intravital epifluorescence-microscopy over 4 h after BD induction; functional capillary density (FCD), leukocyte adherence (AL) in post-capillary venules, histology and pancreatic enzymes were investigated. Four hours after BD, FCD decreased (333 +/- 11 vs. baseline 444 cm/cm2 +/- 5 SEM; p<0.01) and showed lower values than CO (388 +/- 9 p<0.01). In BD, AL was increased (628 cells/mm2 +/- 110 SEM vs. baseline 123 +/- 32, and vs. CO 180 +/- 33; p<0.001). BD caused increased histological damage (CO 1.6 score-points +/- 0.7 SD vs. BD 8.3 +/- 7.1; p<0.05). Amylase was higher in BD (p<0.05) but did not reach pathological values. We show for the first time that BD causes relevant changes in pancreatic microcirculation, histology and leukocyte endothelial interaction which might have a serious impact on the function of grafts. New strategies for preventing this damage are therefore highly desirable in order to improve the outcome of pancreas transplantation.     

Influence of nitric oxide on microcirculation in pancreatic ischemia/reperfusion injury: an intravital microscopic study

Recently, protective effects of nitric oxide donors in pancreatic ischemia/reperfusion (IRI) injury have been described. Their role in post-ischemic microcirculation was previously not investigated. Ischemia reperfusion was induced in an isolated pancreatic tail segment in situ. Animals were randomized to four experimental groups (n=7 animals/group), the control group (CO) received saline as placebo. Treatment groups received either sodium nitroprusside (SN) 5 min before until 2 h after reperfusion, l-arginine (LA) 30 min before reperfusion until 2 h after reperfusion or sodium nitroprusside and l-arginine (SNLA) together. After induction of ischemia (2 h) post-ischemic microcirculation was observed for 2 h by intravital-fluorescence microscopy. Functional-capillary density (FCD), leukocyte adherence in post-capillary venules (LAV) and histological damage were analysed. After reperfusion FCD decreased in all groups (P<0.05). FCD was significantly restored in all groups with administration of nitric oxide donors after reperfusion (P<0.05) as compared to CO without significant difference between the individual nitric oxide donor groups. Leukocyte adherence was significantly increased 1 h and 2 h after reperfusion (P<0.001) as compared to baseline, which was lower in all nitric oxide donor groups. Histological damage in the pancreatic tail-segment was significantly reduced in nitric oxide donor groups (P<0.01). Administration of nitric oxide donors might be useful in ischemia-reperfusion injury of the pancreas by its protective effect on microcirculation and inflammatory reaction.     

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.     

Prevention of lethal murine pancreas ischemia reperfusion injury is specific for tetrahydrobiopterin

Tetrahydrobiopterin has been shown to efficiently abrogate ischemia reperfusion injury (IRI). However, it is unclear, whether its beneficial action relies on cofactor activity of one of the five known tetrahydrobiopterin-dependent reactions or on its antioxidative capacity. We therefore compared tetrahydrobiopterin with the pterin derivate tetrahydroneopterin (similar biochemical properties, but no nitric oxide synthase cofactor activity) and the antioxidants vitamin C and 5-methyltetrahydrofolate. Donor mice were pretreated with tetrahydrobiopterin, tetrahydroneopterin, vitamin C, or 5-methyltetrahydrofolate. Pancreatic grafts were subjected to 16-h cold ischemia time and implanted in syngeneic recipients. Untreated and nontransplanted animals served as controls. Following 2-h reperfusion, microcirculation was analyzed by intravital fluorescence microscopy. Graft damage was assessed by histology and nitrotyrosine immunostaining, and tetrahydrobiopterin levels were determined by HPLC. Recipient survival served as ultimate readout. Prolonged cold ischemia time resulted in microcirculatory breakdown. Only tetrahydrobiopterin pretreatment succeeded to preserve the capillary net, whereas all other compounds showed no beneficial effects. Along with increased intragraft tetrahydrobiopterin levels during recovery and implantation, only tetrahydrobiopterin pretreatment led to significant reduction of IRI-related parenchymal damage enabling recipient survival. These results show a striking superiority of tetrahydrobiopterin in preventing lethal IRI compared with related compounds and suggest nitric oxide synthases as treatment target.     

ET-1 induces pancreatitis-like microvascular deterioration and acinar cell injury.

Using in vivo microscopy red blood cell (RBC) velocities, functional capillary density (FCD) and capillary diameters were estimated after inducing acute pancreatitis by intraductal infusion of sodium taurocholate (0.8 ml; 4%) or after topical superfusion of the pancreas with ET-1 (100 pmol). Sodium taurocholate mediated a significant decrease in RBC velocities between 50 and 70%, transient decrease in capillary diameters by 10%, and a sustained decrease in FCD between 60 and 70% paralleled by a dramatic heterogeneity in blood flow. Topical superfusion of the exteriorized pancreas with ET-1 caused a significant decrease in RBC velocities between 65 and 75%, a sustained decrease in capillary diameters by 10%, and a decrease in FCD by 45% accompanied by an increase in flow heterogeneity. Following sodium taurocholate infusion pancreas histology revealed a severe edema and sublobular acinar cell necrosis, while topical ET-1 application displayed a severe edema of the pancreas with focal acinar cell necrosis. Thus, ET-1 mediated a deterioration of the pancreatic microcirculation, which is similar to the microcirculatory failure found in sodium taurocholate-induced experimental pancreatitis and was associated with focal acinar cell necrosis. We are thus inclined to hypothesize that endothelin released by injured endothelial cells during acute biliary pancreatitis promotes microcirculatory failure and ischemia in acute pancreatitis, eventually leading to acinar cell necrosis.     

Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function

Ischemia-reperfusion injury (IRI) results in increased rates of delayed graft function and early graft loss. It has recently been reported that hydrogen sulfide (H₂S) protects organ grafts against prolonged IRI. Here, we investigated whether the preservation of pancreas in University of Wisconsin (UW) solution supplemented with AP39, which is a mitochondrial-targeted H₂S donor, protected pancreatic islets against IRI and improved islet function. Porcine pancreata were preserved in the UW solution with AP39 (UW + AP39) or the vehicle (UW) for 18 h, followed by islet isolation. The islet yields before and after purification were significantly higher in the UW + AP39 group than in the UW group. The islets isolated from the pancreas preserved in UW + AP39 exhibited significantly decreased levels of reactive oxygen species (ROS) production and a significantly increased mitochondrial membrane potential as compared to the islets isolated from the pancreas preserved in the vehicle. We found that the pancreas preserved in UW + AP39 improved the outcome of islet transplantation in streptozotocin-induced diabetic mice. These results suggest that the preservation of pancreas in UW + AP39 protects the islet grafts against IRI and could thus serve as a novel clinical strategy for improving islet transplantation outcomes.     

Acute effects of local cold therapy in superficial burns on pain,in vivo microcirculation,edema formation and histomorphology

Background

Local cold therapy for burns is generally recommended to relief pain and limit tissue damage, however, there is limited data of its physiological benefit. This study aimed to evaluate pathophysiological effects of cold therapy in superficial burn on microcirculation, edema formation, and histomorphology.

Methods

In 12 volunteers (8f, 4m; aged 30.4 ± 14.1 years) circumscribed superficial burn was induced on both hand back and either left untreated as control (control-group) or treated by local-cold-application (cold-treatment-group). Prior to burn (t0), immediately (t1), 15 min (t2), and 30 min (t3) following cold therapy, following parameter was evaluated using intravital-microscopy; epidermal-thickness (ET), granular-cell-size (GCS), individual-blood-cell-flow (IBCF), and functional-capillary-density (FCD).

Results

Both ET and GCS increased significantly more in control-group and slightly in cold-treatment-group in t1, while turns to insignificant t2 onwards. IBCF and FCD raised up in control-group compared to dramatically decrease in cold-treatment-group in t1. In t2 both parameter remains in control-group and increased in cold-treatment-group. Comparison of both groups for IBCF and FCD indicates significant difference in t1 and t2, however, insignificant in t0 and t3.

Conclusions

Microcirculation, edema formation, and histomorphology of superficial burn has been significantly influenced through immediate cold therapy, however, this alterations are transient and turns to ineffective after 30 min.     

Hemodynamic effects of inducible nitric oxide synthase and nitrotyrosine generation in heart failure.

OBJECTIVES: The hemodynamic effects of cardiac inducible nitric oxide synthase (iNOS) and of iNOS-mediated peroxynitrite in patients with left ventricular (LV) dysfunction are unclear. The present study investigates the incidence and functional significance of iNOS expression and nitrotyrosine formation in patients with heart failure. METHODS: LV endomyocardial biopsies obtained from 24 patients with heart failure due to idiopathic dilated cardiomyopathy (ejection fraction [EF] <45% and left ventricular end-diastolic volume index [LVEDVI] >102 ml/m2) were analyzed for iNOS and nitrotyrosine. LV contractile performance was assessed by left ventricular ejection fraction (LVEF) and stroke work normalized for end-diastolic pressure (SW/EDP). LV filling pattern was assessed by Doppler E/A wave ratio, deceleration time (DT) of early LV filling and indexed LV end-diastolic volume normalized for EDP as a marker of diastolic distensibility. RESULTS: iNOS immunostaining correlated significantly with nitrotyrosine formation (r = 0.86, p < 0.001). In the whole study group, patients expressing iNOS (n = 13) showed larger LV end-diastolic (173 +/-16 vs 128 +/- 9 ml/m2, p = 0.031) and end-systolic volume indices (110 +/- 16 vs 61 +/- 9 ml/m2, p = 0.018) and similar LVEDP (18 +/- 2 vs 21 +/- 2 mm Hg, p = 0.227). In patients with advanced heart failure and reduced pre-load reserve (LVEDP > 16 mm Hg, n = 18), iNOS protein and nitrotyrosine formation correlated positively with LVSW/EDP (r = 0.65, p = 0.03 and r = 0.64, p = 0.04, respectively), DT (r = 0.96, p < 0.01 and r = 0.88, p < 0.01, respectively) and inversely with E/A (r = -0.82, p < 0.01 and r = -0.88, p < 0.01, respectively). In addition, nitrotyrosine formation correlated positively with LVEDVI/EDP (r = 0.64, p = 0.02). Advanced iNOS-positive heart failure patients had a higher LVEDVI/EDP compared with iNOS-negative patients (5.30 +/- 0.64 vs 3.13 +/- 0.34 ml/mm Hg x m2, p = 0.02). CONCLUSIONS: In heart failure, iNOS protein expression is associated with nitrotyrosine formation. Although iNOS-positive patients are generally characterized by larger LV volume and depressed function, the preserved NO generation appears to be associated with higher cardiac work due to the preserved Frank-Starling relationship in end-stage heart failure.     

Influence of tissue nitration on tissue damage with thermal injury

A gas mediator, nitric oxide, is converted into peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of tyrosine residue in protein and enhances the inflammatory response in the prolonged phase. In this study, the authors investigated the relationship between peroxynitrite-mediated tissue nitration and tissue damage with thermal injury using an experimental burn model. The content of nitrotyrosine in the burned tissue significantly increased 1 to 6 h after injury. The nitrotyrosine content in the burned ear significantly decreased with 100 mg/kg of LNAME administration. Vascular hyperpermeability was also significantly suppressed in the iNOS antibody immunoneutralized mice 6 h after injury. There was a positive correlation between the severity of tissue damage, an indicator of which is the increase in the weight of the burned ear along with the development of edema after injury, and the concentration of nitrotyrosine in the wound tissues. Nitrotyrosine-like immune reactants were also diffusely detected in the burned region and the surrounding areas. These results indicate that peroxynitrite is produced in the surrounding burned region and a reaction of nitration of tissue tyrosine is involved with tissue damage at the burn wound. Therefore, to prevent the systemic vascular hyperpermeability and tissue damage in a large area burn or severe burn patients, the administration of NOS inhibitors or radical erasers may be easy to manage generally by inhibition of peroxynitrite formation.     

Can the preprocurement pancreas suitability score predict ischemia-reperfusion injury and graft survival after pancreas transplantation?

Background

Ischemia-reperfusion injury (IRI) is common after pancreas transplantation, leading to pancreatitis or thrombosis with the need for relaparotomy or even graft loss. Optimal donor selection may reduce the postoperative morbidity of IRI. The Eurotransplant preprocurement pancreas suitability score (P-PASS) seeks to identify ideal donors with a value <17. Owing to the organ shortage the waiting time for pancreas transplantation is increasing, a problem that may be addressed with the use of extended-criteria donors. We analyzed our pancreas transplantations regarding postoperative complications according to the P-PASS. To reflect IRI we used the peak C-reactive protein (CRP) levels during the first 3 postoperative days.

Methods

From January 2009 to July 2010, we transplanted 52 pancreas grafts, including, 50 simultaneous pancreas-kidney transplantations (SPK), 1 after a kidney graft, and 1 alone. For 3 SPK donors the P-PASS was not available. All transplantations were performed using systemic venous and enteric drainage. The immunosuppression protocol included antibody induction with antithymocyte globulin and maintenance therapy with steroids, tacrolimus, and mycophenolate mofetil. The peak CRP in the first 3 postoperative days was used as a marker for IRI.

Results

The mean P-PASS of our donors was 16.4 ± 2.6 (range, 12–22). We compared 24 patients receiving organs from “ideal” donors (P-PASS <17; ID) with 25 receiving grafts from extended-criteria donors (P-PASS ≥17; ED). There was no significant difference in the incidence of graft loss among ID versus ED grafts (20.8% vs 20.0%; P = 1.0). Comparing the rates of postoperative complications of patients, we did not observe a significant difference in graft thrombosis (4.2% vs 16.0%; P = .349), relaparotomy (29.2% vs 40.0%; P = .551), a pancreatic fistula (37.5% vs 28.0%; P = .543), or the length of hospital stay (36.5 ± 19.2 vs 37.4 ± 20.8 days; P = .875), respectively. Regarding IRI, there was no significant difference in peak CRP values (14.1 ± 5.5 vs 16.2 ± 6.0 mg/dL; P = .211).

Conclusion

This single center analysis failed to show that P-PASS significantly predicted pancreas graft survival, postoperative morbidity, or IRI severity. These findings suggested a chance to increase the donor pool using extended-criteria donors.     

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.     

Improving pancreas graft utilization through importation

Background

We analyze our outcomes utilizing imported allografts as a strategy to shorten wait list time for pancreas transplantation.

Methods

This is an observational retrospective cohort of 26 recipients who received either a locally procured (n = 16) or an imported pancreas graft (n = 10) at our center between January 2014 and May 2017. Wait list times of this cohort were compared to UNOS Region 9 (New York State and Western Vermont). Hospital financial data were also reviewed to analyze the cost‐effectiveness of this strategy.

Results

Imported pancreas grafts had significantly increased cold ischemia times (CIT) and peak lipase (PL) levels compared to locally procured grafts (CIT 827 vs 497 minutes; P = .001, PL 563 vs 157 u/L; P = .023, respectively). There were no differences in graft or patient survival. The median wait time was significantly lower for simultaneous kidney‐pancreas transplants at our center (518 days, n = 21) compared to Region 9 (1001 days, n = 65) P = .038. Despite financial concerns, the cost of transport for imported grafts was offset by lower standard acquisition costs.

Conclusions

Imported pancreas grafts may be a cost‐effective strategy to increase organ utilization and shorten wait times in regions with longer waiting times.     

缺血-再灌注损伤对大鼠急性胰腺炎胰腺细胞凋亡的影响

目的探讨缺血一再灌注(I／R)损伤对大鼠急性胰腺炎(AP)胰腺细胞凋亡的影响。方法将SD大鼠54只按编号法随机分为对照组(n=6)、胰腺炎组(n=24)和I／R损伤组(n=24)。经胆胰管逆行加压注入3％牛磺胆酸钠建立大鼠AP模型，在此基础上，I／R损伤组通过暂时阻断脾下动脉造成局部胰腺I／R模型，对照组于术后lh，其余两组于术后1h、3h、6h和12h采取断颈方法分批处死动物，应用末端脱氧核苷酸转换酶(TdT)介导的原位末端标记(TUNEL)法检测缺血一再灌注区胰腺细胞凋亡。并观察其病理改变。结果胰腺炎组大鼠术后1h、3h胰腺组织仅为充血、水肿性改变，6h出现出血、坏死性改变；而1／R损伤组大鼠术后1h缺血一再灌注区胰腺呈现出血、坏死性改变，病变持续加重；AP后胰腺凋亡细胞明显增多，I／R损伤组和胰腺炎组的凋亡细胞高峰值分别在术后3h和6h；I／R损伤组术后1h、3h缺血一再灌注区胰腺凋亡细胞显著高于相应时相的胰腺炎组(P＜0．01，P＜0．05)．而6h、12h明显低于胰腺炎组(P＜O．05，P＜O．01)。结论I／R损伤在促发胰腺炎从水肿型向出血坏死型转化过程中，同时诱导胰腺细胞凋亡，细胞凋亡可能是阻止AP病变加重的一个有利反应。