The dual role of tetrahydrobiopterin in the prevention of early parenchymal damage following murine pancreas transplantation

Abstract

Background: Tetrahydrobiopterin (H4B) is both an essential co-factor of nitric oxide synthases (NOS) and a strong antioxidant. Under oxidative stress-associated H4B depletion NOS is known to produce oxygen radicals rather than nitric oxide (NO) by uncoupling, a phenomenon thought to contribute to endothelial dysfunction in a variety of vascular diseases. Recent findings showed also dramatically reduced ischemia/reperfusion injury (IRI) following murine pancreas transplantation after H4B substitution. To differentiate the antioxidant effect from uncoupling-prevention we compared in this study H4B supplementation previous to organ retrieval with supplementation of tetrahydroneopterin (H4N), a pterin with equal antioxidant potential but no co-factor activity. Material and Methods: Male syngeneic C57BL6 (H-2b) mice, 10–12 weeks old, were used as size-matched donor and recipient pairs. Murine cervical vascularized pancreas transplantation was performed with a modified no-touch technique. Pancreatic grafts were subjected to 16h prolonged cold ischemia time (CIT) as well as to 45 min warm ischemia time (WIT) and different treatment regiments: untreated animals (I), H4B 50 mg/kg i.m. prior to organ retrieval (II) and H4N 50 mg/kg i.m. prior to organ retrieval (III). Non transplanted animals served as controls (IV). After 2h of reperfusion intravital fluorescence microscopy was used for analysis of graft microcirculation by means of functional capillary density (FCD) and capillary diameters (CD). Quantitative assessment of parenchymal damage was analyzed by histology (H&E) and by performing nitrotyrosine-immunostaining to determine peroxynitrite formation. Intragraft pteridin levels were measured by HPLC. Results: Following prolonged CIT and 45 minutes of WIT only pancreatic grafts treated with H4B prior to organ explantation (II) displayed markedly higher values of FCD and CD compared to non treated animals (I) (p<0.01 and p<0.05 respectively). In contranst, pre-treatment of donor animals with H4N did improve neither FCD nor CD levels. Compared to non treated animals H4B and H4N pre-treatment significantly attenuated peroxynitrite formation (p<0.05). However, parenchymal damage like acinar, fat and hemorrhagic necroses after after 2 h graft reperfusion were significantly reduced only by H4B pre-treatment (p<0.05). Partially oxidized H4B was substantially decreased in the H4B treatment group. Conclusions: The protective effect of H4B donor pre-treatment may rely not only on its antioxidative activity but also on its NOS co-factor capacity and might therefore be a novel, promising agent in clinical pancreas transplantation preventing early parenchymal damage after graft reperfusion.