Experimental Pancreas Transplantation Research Articles

Breaking the limits of experimental pancreas transplantation: Working toward the clinical ideal graft.

Pancreas transplantation is, at present, the only curative treatment for type-1 diabetes that maintains normoglycemia thus avoiding complications arising from poor glycemic control. Despite its great benefits, the number of pancreas transplants has decreased significantly since its inception in the late 1960s, largely due to demographic changes and the consequent suboptimal quality of donors. The selection criteria for pancreas donors mainly depend on morphological variables such as fatty infiltration, fibrosis, or edema, as well as both functional (amylase and lipase) and clinical variables of the donor. However, the final criterion in the decision-making process is the somewhat subjective assessment of a trained surgeon. That being said, the recent incorporation of graft perfusion machines into clinical practice seems to be changing the work dynamics of the donor organ retrieval team, facilitating decision-making based on objective morphological and functional criteria. Normothermic perfusion using perfusate with supplemental oxygen replicates near physiological parameters thus being a promising strategy for organ preservation. Nevertheless, optimum perfusion parameters are difficult to establish in pancreas transplantation given its complex vascular anatomy combined with an intrinsically low blood flow. The objective of this work is to analyze the results published in the recent literature relating to the considerations of ex-vivo normothermic graft perfusion machines and their usefulness in the field of pancreas transplantation.

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.

Altered cellular membrane fluidity levels and lipid peroxidation during experimental pancreas transplantation

Although the pathogenesis of ischemia reperfusion (IR) injury is based on complex mechanisms, free radicals play a central role. We evaluated membrane fluidity and lipid peroxidation during pancreas transplantation (PT) performed in 12 pigs (six donors and six recipients). Fluidity was measured by fluorescence spectroscopy, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations were used as an index of lipid oxidation. Pancreatic tissues were collected as follows: (A) donor, immediately before vascular clamping; (B) graft, following perfusion lavage with University of Wisconsin preservation fluid; (C) graft, after 16h of cold ischemia; and (D) recipient, 30min vascular postreperfusion. Fluidity and MDA and 4-HDA concentrations were similar in cases A, B, and C. However, there was significant membrane rigidity and increased lipid peroxidation after reperfusion (D). These findings suggest that reperfusion exaggerates oxidative damage and may account for the rigidity in the membranes of allografts during PT.

Effects of Premedication With Tiletamine/Zolazepam/Medetomidine During General Anesthesia Using Sevoflurane/Fentanyl in Swine Undergoing Pancreas Transplantation

Objective To assess cardiac and hemodynamic responses and body temperature during long-term general anesthesia using sevoflurane/fentanyl after premedication with a tiletamine/zolazepam/medetomidine combination in swine undergoing experimental pancreas transplantation. Materials and methods Twelve Landrace female pigs of means weight 46.4 ± 5.1 kg were premedicated by intramuscular administration of tiletamine/zolazepam (3.5 mg/kg), medetomidine (0.03 mg/kg), and atropine (0.02 mg/kg), before anesthesia with 0.75 minimum alveolar concentration sevoflurane and continuous intravenous fentanyl infusion (5.7 ± 0.7 μg/kg/h). Assessment of heart rate, arterial blood pressure, and temperature in pigs undergoing allogenic pancreas transplant surgery were registered at the start of anesthesia (T0), as well as at 60 (T60), 120 (T120), and 180 (T180) minutes after T0, and finally at the end of anesthesia (T anesthesia end), when we switched off the sevoflurane vaporizer. Analysis of variance was used to determine differences between times with P < .05 considered significant. Results are given as mean values ± standard deviations. Results Arterial blood pressure significantly decreased from T120 to the end of anesthesia, while a significantly decreased heart rate was only evident at T60. Body temperature decreased significantly from T60 to the end of anesthesia. These decreases, however, lacked clinical relevance; all parameters were within normal range. No major anesthetic complications were observed in this study. Conclusions The administration of a tiletamine/zolazepam/medetomidine combination as premedication in swine subjected to pancreas transplantation allowed for a safe reduction of sevoflurane/fentanyl requirements during long-term general anesthesia. Despite arterial blood pressure and body temperature evidencing a decrease during anesthetic maintenance, all parameters remained within normal range values.

In vivoquantification of oxygen-free radical release in experimental pancreas transplantation

Reactive oxygen species (ROS) were drawn to the attention in the setting of organ transplantation when the 'injury hypothesis' postulated a link between oxidative stress and the activation of the innate immunity of the recipient. While the occurrence of ROS during organ transplantation is undoubted, their onset and magnitude remain largely unknown. We therefore measured ROS using a novel cyclic hydroxylamine spin probe CMH (1-hydroxy-3- methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) during syngeneic experimental pancreas transplantation in rats in vivo. Organs were subjected to two different cold preservation methods [University of Wisconsin preservation solution (UW) or normal saline] for 18 h. During the first 90 min of reperfusion, samples were collected and analysed using electron paramagnetic resonance signalling. Isolated blood-free perfused organs (IPO) were used for comparison. Analysis showed that it is feasible to detect ROS using CMH spin probes. While IPO organs displayed a very early ROS release, there was no ROS increase in the UW preserved group compared to NaCl. These findings were in line with conventional markers of organ damage such as serum lactate, glucose, potassium as well as tissue ATP levels. CMH spin probes might become a useful tool for the in vivo animal testing of antioxidative substances in models of solid organ transplantation.

Induction of Heme Oxygenase-1 Improves the Survival of Pancreas Grafts by Prevention of Pancreatitis After Transplantation

Ischemia/reperfusion (I/R) injury after pancreas transplantation might result in graft pancreatitis. The role of heme oxygenase-1 (HO-1) in pancreas transplantation and prevention of graft pancreatitis is unknown. We studied the impact of HO-1 induction with cobalt protoporphyrin (CoPP) in experimental pancreas transplantation with moderate (6 hr) and prolonged (20 hr) cold ischemic time (CIT). Donor animals received CoPP 5 mg/kg intraperitoneal at 48 hr or intraperitoneal saline injections in the corresponding control groups before procurement. Harvested grafts were perfused with HTK solution and stored at 4 degrees C. After prolonged CIT, graft survival was 100% with CoPP pretreatment in contrast to only 37.5% without pretreatment. CoPP-pretreated grafts demonstrated an unimpaired endocrine graft function at moderate and prolonged CIT. Serum lipase activity as a sign of exocrine preservation was significantly lower. In addition, morphological architecture was well preserved. CoPP pretreatment markedly increased HO-1 gene expression in donor pancreas (130-fold increase) by means of quantitative reverse transcriptase -polymerase chain reaction. Immunohistochemical examinations showed that the increase of HO-1 on the protein level was related to HO-1-positive donor macrophages in the pancreas grafts. HO-1 overexpression was accompanied by significant decrease of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-2, IL-6, interferon-y, and by significant increase of the anti-inflammatory cytokine IL-10 and less expression of adhesion molecules such as e- and p-selectins. HO-1 is highly inducible in the allograft rat pancreas and associated with a survival benefit and good graft function after transplantation. This study contributes to the beneficial potentials of HO-1 for the prevention of graft pancreatitis.

Characterization of Ischemia/Reperfusion–Induced Gene Expression in Experimental Pancreas Transplantation

The aim of this study was to identify genes that are differentially expressed in the early period after pancreatic cold ischemia/reperfusion (I/R) injury. Grafts of isogeneic rat pancreaticoduodenal transplantation were subjected to different preservation solutions and cold ischemia times (CITs): University of Wisconsin (UW), 6-hour CIT; UW, 18-hour CIT; and physiologic saline solution, 6-hour CIT. Animals that did not receive transplants served as controls. At 2-hour reperfusion, grafts were removed and pancreatic RNA was isolated, pooled, and hybridized to Affymetrix RG-U34A arrays. Quantitative reverse-transcription polymerase chain reaction was used to confirm the results of microarray technology. A total of 49 genes were consistently upregulated (more than threefold) in all three groups of transplant recipient animals. Prominent genes include transcription factors; cytoskeletal factors; heat-shock proteins (e.g. Hsp27, Hsp90); molecules involved in inflammation (e.g. PAPIII), immunology, signal transduction, and translation; and genes that have not been associated with I/R injury so far (e.g. Best5). Messenger RNA levels of some genes were exclusively downregulated in response to the different conditions applied to the pancreatic grafts: Cybb, Reg3a, Per2, BMAL1, MAP, and Isl2. These results provide new insight in I/R-induced gene expression after experimental pancreas transplantation. The reported upregulation of heat shock proteins, Best5, and PAPIII may play a pathologic role in pancreatic cold I/R injury and could therefore provide a promising perspective for further investigations.

Changes of inducible protein-10 and regulated upon activation, normal T cell expressed and secreted protein in acute rejection of pancreas transplantation in rats

To investigate the role of IFN-gamma inducible protein -10 (IP-10) and regulated upon activation, normal T cell expressed and secreted (RANTES) protein in acute pancreatic allograft rejection in rats. An experimental pancreas transplantation model was established using diabetic SD rats as the recipient, induced by applying streptozocin (STZ). Pancreas transplantation was performed with a physiologic method of portal venous and enteric drainage. Rats were divided into two groups, isograft group (group A, n = 24) and allograft group (group B, n = 24) in which either healthy SD rats or Wistar rats served as donors, respectively. Twelve diabetic or healthy SD rats were used as controls. At d 1, 4, 7, and 10 post transplantation, serum IP-10 and RANTES were assessed by ELISA and their expression in the allografts was determined by immunohistochemistry. In group B (allograft group), the development of acute rejection was significantly correlated with increased serum concentration and tissue expression of IP-10 and RANTES, with a peak level at d 7 post transplantation. In contrast, there was no obvious change before and after transplantation in group A (isograft group). Our study suggests a possible role of IP-10 and RANTES in acute rejection and early monitoring of chemokines may be helpful in predicting the outcome of pancreas transplantation.

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

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

Reduction of ischemia/reperfusion injury by antithrombin III after experimental pancreas transplantation

Background Graft pancreatitis is a major complication after pancreas transplantation. Antithrombin III (AT III) is an anticoagulatory and anti-inflammatory substance. The aim of our study was to evaluate a prophylactic application of AT III in experimental pancreas transplantation. Methods Pancreas transplantation was performed in rats. Cold ischemia time (University of Wisconsin solution at 4°C) was 12 hours. After 4 hours of reperfusion, pancreatic enzymes were assessed and the pancreas was evaluated by intravital microscopy and histologic and immunohistochemical examination. Recipients were allocated randomly to 2 groups: 1 control group (n = 6) and 1 group in which recipients received 125 IU AT III/kg 30 minutes before reperfusion (n = 6). Six animals that did not undergo transplantation served as healthy controls. Results Enzyme levels showed no differences between the 2 transplantation groups but were significantly ( P <.05) higher than in the control group. Histologic damage was significantly less evident in animals that received AT III compared with transplantation animals that did not receive AT III. During intravital microscopy, animals receiving AT III showed significantly higher capillary and venular erythrocyte velocities compared with untreated transplantation animals. The leukocyte-endothelium interaction in postcapillary venules was decreased significantly in animals with AT III treatment. Conclusions AT III pretreatment decreases tissue damage by attenuating microcirculatory disturbances and leukocyte adherence in experimental graft pancreatitis by its anti-inflammatory and anticoagulatory properties.

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

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

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

Abstract 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.

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 degrees 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.

Pathomorphologic and microcirculatory changes and endothelin-1 expression in UW-and Celsior-preserved pancreata in experimental pancreas transplantation

Pathomorphologic and microcirculatory changes and endothelin-1 expression in UW-and Celsior-preserved pancreata in experimental pancreas transplantation

Ischemia/reperfusion injury induces acinar cell apoptosis in experimental pancreas transplantation

Ischemia/reperfusion injury induces acinar cell apoptosis in experimental pancreas transplantation

Comparison of Celsior and UW Solution in Experimental Pancreas Preservation

Background. The University of Wisconsin solution (UW) is the gold standard for pancreas preservation. Celsior (CEL) was formulated specifically for heart preservation. Recently, experimental and clinical experience has been reported on the application of CEL to abdominal organs. In this animal study, pancreas preservation with CEL was compared with that in UW solution.Patients and Materials. Heterotopic, allogeneic pancreaticoduodenal transplantation was performed in female Göttingen Minipigs (n = 12 donors, n = 12 recipients). The grafts were flushed and stored for 6 h at 4°C in UW or CEL. The recipients were randomized into two groups receiving either UW (n = 6)- or CEL (n = 6)-preserved grafts with a follow-up of 5 days. Blood flow (laser Doppler), partial oxygen tension, histological changes, endothelin-1 (plasma, immunohistochemistry), lipase, amylase, trypsinogen activation peptide, and C-reactive protein (CRP) were measured.Results. Partial oxygen tension was lower in the CEL group (P < 0.05). However, blood flow did not differ between UW- and CEL-preserved organs. The histomorphologic analysis of the pancreatic grafts revealed significantly less edema in the UW-preserved organs. Serum levels of amylase, lipase, CRP, and TAP taken from the central venous blood were comparable in the two groups, except for higher amylase values 36 h after reperfusion in the CEL group compared to the UW group (P < 0.05). Likewise, TAP taken from the portal venous effluent of the graft was found to be higher in the CEL group than in UW (P < 0.05). Endothelin-1 serum levels rose significantly during reperfusion without differences between the two groups. ET-1 immunohistochemistry revealed increased local ET-1 during reperfusion in all grafts. However, the ET-1 immunostaining in the CEL group was more pronounced than that in the UW group (P < 0.05).Conclusions. Our results suggest that CEL solution is not as effective in preventing pancreatic ischemia/reperfusion damage as the standard UW solution in experimental pancreas transplantation. Increased ET-1 immunostaining and reduced ptiO2 in the CEL group indicate increased microcirculatory damage in the CEL group.

High levels of nitric oxide metabolites can be correlated with rejection episodes in experimental pancreas transplantation

High levels of nitric oxide metabolites can be correlated with rejection episodes in experimental pancreas transplantation

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.

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.