damage. It 169939-93-9 describes four categories: edema, acinar necrosis, haemorrhage and fat necrosis, and inflammatory infiltrates. Statistics Results are expressed as mean 6 standard error of the mean. Statistical analysis was performed using GraphPad Prism 5. Kruskal-Wallis test was used if multiple groups were compared. If statistical significance was achieved, all pairs were compared among each other using the Mann-Whitney-U-test and the Bonferroni post-test. Kaplan-Meier curve was used for survival analyses and groups were compared using the log rank test. A p value of,0.05 was considered to be statistically significant. Immunohistochemistry Paraffin was removed from cut tissue sections by heating them in citrate buffer, pH 6.0. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide and finally washed with Dulbecco’s phosphate buffered saline. Nitrotyrosine-IHC was performed in a diaminobenzidinetetrahydrochloride autostainer, using an anti-nitrotyrosine rat polyclonal antibody from Upstate Biotechnology at 1:100 dilution. For staining, secondary antibody peroxidase-labeled polymer and 3,39 DAKO were used. Haemalaun was used for counterstaining. For quantification purposes, the product of proportion of positive cells in quartiles, and the staining intensity was calculated, yielding a total semiquantitative immunostaining score ranging from 0 to 12. Results Effect of mouse donor genotype and BH4 treatment on early microcirculatory damage First, we investigated dependence of microcirculatory alterations on donor genotype and BH4 treatment. As depicted in figure 1, representative intravital fluorescence images of 
nontransplanted pancreatic tissues looked comparable independently from genotype. Prolonged CIT resulted in a marked breakdown of the microcirculation in untreated, transplanted wt, eNOS2/2 and iNOS2/2 grafts. In contrast, following prolonged CIT grafts lacking the neuronal isoform displayed a regular capillary mesh comparable PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19689163 to non-transplanted tissue. Donor pre-treatment with BH4 prevented the capillary breakdown independently of the genotype, except for nNOS2/2 where no further improvement in the already wellperfused grafts could be observed. Mean FCD value in non-transplanted organs was 219.07619.44 in wt and did not differ from knockout strains. Exogenous BH4 application did not influence microcirculation of non-transplanted pancreata. In contrast, prolonged CIT followed by 2 hours reperfusion resulted in a breakdown of the microcirculation in untreated wt, eNOS2/2 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692147 as well as iNOS2/2 grafts, consistently displaying lower FCD values compared to the respective treated groups, without however reaching statistical significance. FCD values further declined following 4 h reperfusion in untreated wt and eNOS2/2 grafts, but not in iNOS2/2 grafts, where FCD values did not differ significantly from the previous time point. At this time point, however, FCD levels were significantly lower compared to treated counterparts. In contrast, nNOS2/2 grafts, both, untreated and treated organs, did not show any decrease in FCD values over the Western Blot To complement IHC findings we additionally performed nitrotyrosine western blots according to a protocol described in our previous publication. Biopterin tissue levels Intragraft BH4 concentrations were obtained by a method modified from Fukushima and Nixon as detailed before. Briefly, tissue was homogenized in 5 mM dithioerythrol and incubated with 20 ml 0.5 M HCl and 0.05 M iodine for acidi