Artikel
Tetrahydrobiopterin protects pancreatic grafts from ischemia-reperfuson-injury: does the endothelial nitric oxide synthase play a role?
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Veröffentlicht: | 20. Mai 2011 |
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Introduction: Ischemia-reperfusion-injury (IRI) following pancreas transplantation (PTX) is a major cause of graft pancreatitis. The essential cofactor of nitric oxide synthase (NOS) and potent antioxidant tetrahydrobiopterin (H4B) was shown to significantly attenuate IRI-induced graft pancreatitis. Since underlying mechanisms are still unclear, we investigated if the eNOS enzyme is the main target using an eNOS-/- model.
Materials and methods: In a heterotopic PTX model syngeneic C57Bl6 mice (wild type and eNOS-/-) served as donor-recipient pairs. Donors of both genotypes were either untreated or treated with 50-mg/kg b.w. H4B i.m. All grafts were subjected to 16h cold ischemia time (CIT). Non-transplanted animals of both genotypes served as controls. Following 15min and 2h reperfusion, microcirculation was analyzed by confocal intravital fluorescence microscopy, parenchymal damage was assessed by H&E histology and nitrotyrosine immunostaining. Following 15min reperfusion, microarray analysis (Affymetrix®) was performed and results were confirmed by RTqPCR (Taqman-technology).
Results: Prolonged CIT resulted in a pronounced microcirculatory breakdown, severe graft pancreatitis, as well as intragraft peroxynitrite formation. As early as 15min following reperfusion no differences between treated and untreated grafts, and between genotypes were observed. After 2h reperfusion time, H4B-treated grafts displayed a consistent amelioration of graft microcirculation (p=0.09). In addition, IRI-induced parenchymal damage and nitrotyrosine formation were reduced in treated grafts, reaching statistical significance only in wild types (p<0.05). Among the complete mouse genome two differently regulated genes (>5-fold) were detected by microarray analysis in grafts reperfused for 15min, and confirmed by RTqPCR. Finally, H4B pre-treatment resulted in a significantly prolonged graft as well as recipient survival, indipendently from the donor genotype (p<0.001).
Conclusion: Comparable intraparenchymal damage of transplanted grafts and similar recipient survival in both genotypes if treated with H4B, suggest, that eNOS is not the major target of H4B in this model.