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. Author manuscript; available in PMC: 2012 Jan 3.
Published in final edited form as: Am J Transplant. 2010 Oct;10(10):2231–2240. doi: 10.1111/j.1600-6143.2010.03262.x

Donor pre-treatment with tetrahydrobiopterin saves pancreatic isografts from ischemia reperfusion injury in a mouse model

M Maglione 1,*,‡, R Oberhuber 1,*, B Cardini 1, K Watschinger 2, M Hermann 3, P Obrist 4, P Hengster 1,3, W Mark 1, S Schneeberger 1, G Werner-Felmayer 2, J Pratschke 1, R Margreiter 1, ER Werner 2, G Brandacher 1
PMCID: PMC3249459  EMSID: UKMS40278  PMID: 20883557

Abstract

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 16h cold ischemia time (CIT) and different treatment regimens: no treatment, 160μM H4B to perfusion solution, H4B 50mg/kg prior to reperfusion and H4B 50mg/kg before organ retrieval. Non-transplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by HPLC.

Compared to non-transplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pre-treatment with H4B resulted in almost completely abrogated IRI-related damage showing graft microcirculation comparable to non-transplanted 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 pre-treatment abrogates ischemia-induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation.

Introduction

Simultaneous kidney pancreas transplantation currently represents the therapy of choice for patients with insulin dependent diabetes mellitus and end-stage renal disease to achieve long-term normoglycemia and insulin independence (1). However, despite several advances in immunosuppressive therapy, organ preservation, surgical techniques, and prophylaxis of infection, ischemia reperfusion injury (IRI), remains a major quest following solid organ transplantation, and in particular following pancreas transplantation (2-5). IRI-associated graft pancreatitis represents a particularly severe complication following pancreas transplantation. It is also a major risk factor for graft thrombosis, which was found to be one of the leading causes of graft loss in the first 6 months posttransplant according to the International Pancreas Transplant Registry (IPTR) (6), (7). Associated pathophysiological events during ischemia and reperfusion are loss of endothelial integrity, expression of pro-inflammatory cytokines and adhesion molecules as well as endothelial activation resulting in microcirculatory disorders (8, 9). A key player involved in this cascade is nitric oxide (NO) generated by nitric oxide synthases (NOS)(10).

(6R)-5,6,7,8-Tetrahydro-L-biopterin (H4B) is an essential co-factor for all three NOS isoforms (endothelial, neuronal and inducible) and thus a critical determinant of NO production. H4B is important for the catalytic activity of the NOS enzymes by stabilizing their active dimeric isoform and by increasing substrate affinity. Furthermore, there is evidence that NOS-bound H4B acts also as scavenger of NOS-derived radicals (11-14).

Oxidative stress generated during various cardiovascular pathologies has been shown to deplete H4B (15, 16). Suboptimal concentrations of H4B then lead to the “uncoupling” of the NOS enzyme resulting in a reduced formation of NO and in an increased reduction of oxygen, generating superoxide anions and hydrogen peroxide and ultimately tissue damage(17).

In a previous study we were able to show that IRI following murine pancreas transplantation was also accompanied by a significant depletion of intragraft H4B concentrations and that the parenchymal damage could be prevented by recipient pre-treatment over 24 hours with H4B (18). However, with deceased donor organs this application is not clinically feasible.

The aim of this study therefore focused on further investigating the therapeutic potential of H4B to reduce IRI-associated parenchymal damage after pancreas transplantation in three different - clinically applicable - treatment regimens: either H4B was added to the perfusion solution, recipients were treated with a single dose of H4B prior to organ reperfusion or donors were pretreated with a single dose of H4B immediately prior to organ retrieval.

Material and Methods

Animals

Ten- to twelve-week-old male C57BL6 (H2b) mice obtained from Harlan-Winkelmann Co. (Borchen, Germany) were used as size-matched donor and recipient pairs. Animals were housed under standard conditions at the animal center of Innsbruck Medical University and given mouse chow and water ad libitum before and after transplantation. All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985), and all experiments were approved by the Austrian Federal Ministry for Education, Arts and Culture.

Pancreas transplantation

Animals were anesthetized with an intramuscular injection of 100mg/kg b.w. ketamine hydrochloride (Ketavet®, Pharmacia GmbH, Germany) and 10-15mg/kg b.w. xylazine (Xylasol®, Dr. E. Gräub AG, Switzerland). All surgical procedures were carried out under clean but not sterile conditions. Briefly, grafts were explanted by sequential separation of the duodenum and the mesenteric axis. Exocrine secretion was managed by ligation of the choledocho-pancreatic duct. Finally, pancreatic grafts were placed in a cervical position with a modified no-touch technique and revascularized using a non-suture cuff technique (19).

Experimental design

All pancreatic grafts were subjected to 16h prolonged cold ischemia (CIT) time. Warm ischemia time (WIT) was strictly standardized at 45 min in all recipient animals (n=5 per group). Perfusion solution used was Custodiol® (HTK, Dr. Franz Köhler Chemie GmbH, Alsbach-Hähnlein, Germany). Study groups consisted of: group I: untreated; group II: H4B-enriched HTK (160μM); group III: H4B 50mg/kg i.m. to the recipient prior to organ reperfusion; group IV: H4B 50mg/kg i.m. to the donor before organ retrieval. Non-transplanted animals served as controls (group V). H4B was obtained from Schircks Laboratories, Jona, Switzerland.

In a second approach, the best treatment arm resulting from the study was analyzed for nitrotyrosine formation; endocrine graft function and recipient survival were compared to untreated animals.

To assess endocrine graft function daily blood glucose was measured in recipients which were treated with a single dose of streptozotocin (Sigma Aldrich, Vienna, Austria) i.p. (312.5mg/kg b.w.) five days prior to transplantation to achieve irreversible hyperglycemia (19). Daily body weight control was performed.

Intravital fluorescence microscopy

Intravital fluorescence microscopy (IVFM) was used for analysis of graft microcirculation 2 hours following reperfusion. To quantify microvascular disorder mean functional capillary density (FCD), defined as the length of all blood cell perfused nutritive capillaries per observation area, and mean capillary diameter (CD), defined as mean value of the three largest capillaries per observation area, were assessed.

In order to enhance the contrast of the microvessels 0.3ml of a 0.4% fluorescein-isothiocyanate (FITC)-labeled dextran (MW 150.000; Sigma Aldrich, Vienna, Austria) were injected via the penile vein. Confocal microscopy was performed with a microlens-enhanced Nipkow disk-based confocal system UltraVIEW RS (Perkin Elmer, Wellesey MA, USA) mounted on the Olympus IX-70 inverse microscope (Olympus, Nagano, Japan). Images were acquired using the UltraVIEW RS software. Each image consists of a z-stack of 20 planes acquired with a 20x objective at a wavelength of 488nm. Selection of observation areas as well as image acquisition was blinded. Quantitative image analysis was done with the PicEd Cora software (Jomesa, Munich, Germany).

Histopathology

For histological examination, grafts were fixed in 10% formaldehyde for 24h, embedded in paraffin, and stained with hematoxylin and eosin (H&E). For quantification purposes the semiquantitative pancreatitis Schmidt score was adopted, which quantifies four categories of parenchymal damage: interstitial edema, acinar necrosis, hemorrhage and fat necrosis and inflammatory infiltrates (20).

Immunohistochemistry (IHC) and Western Blot

For assessment of oxidative parenchymal damage nitrotyrosine–IHC was performed. Graft sections (4μm) were cut from paraffin blocks, mounted on slips and the paraffin removed by heating in citrate buffer, pH 6.0. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide. Nitrotyrosine-IHC was then performed in a diamonbenzidinetetrahydrochloride (DAB) autostainer (DAKO, Copenhagen, Denmark), using an anti-nitrotyrosine rat polyclonal antibody from Upstate Biotechnology (Lake Placid, NY, USA) at 1:100 dilution. For staining, secondary antibody peroxidase-labeled polymer and 3,3′ DAKO were used. Haemalaun was used for counterstaining. For quantification purposes, the product of proportion of positive cells in quartiles (0, 1, 2, 3, 4), and the staining intensity (0 no staining; 1 weak; 2 moderate; 3 strong) was calculated, yielding a total semiquantitative immunostaining score ranging from 0 to 12.

For Western blotting pancreatic tissue was homogenized in 5mM dithioerythritol and 1mM phenylmethylsulfonylfluoride using an ultraturrax mixer (IKA, Germany). Proteins were separated by SDS-Page (12%) and transferred to nitrocellulose membranes (Protran BA 85, Whatman GmbH, Dassel Germany). Membranes were incubated in blocking solution (Dulbecco’s PBS containing 3% (w/v) BSA and 0.1% (v/v) Tween 20). Membranes were incubated with mouse monoclonal [2A12] antibody to 3-nitrotysosine (ab52309, Abcam, Cambridge, UK) diluted 1:1.000 in blocking solution overnight at 7°C. After three 10 min washes in washing buffer (Dulbecco’s PBS containing 0.1% (v/v) Tween 20) membranes were incubated with anti-mouse IgG-horseradish peroxidase conjugate (Sigma Aldrich, Vienna, Austria) diluted 1:10.000 in blocking solution. Blots were shaken for 7 min in ECL® Plus detection reagent (Amersham Biosciences, Freiburg, Germany) and scanned in a Typhoon scanner. Bands were quantified using ImageQuant TL software (Amersham Biosciences, Freiburg, Germany). Signals were related to staining of α-actin following the same staining protocol using mouse monoclonal anti-actin antibody (1:10.000, MAB1501, Millipore, Vienna, Austria) and anti-mouse IgG-horseradish peroxidase conjugate (1:10.000, Sigma Aldrich, Vienna, Autria) to control for protein loading.

H4B tissue levels

To determine intragraft H4B concentrations, tissue was homogenized on ice with an ultraturrax in distilled water containing 5mM dithioerythrol, centrifuged at 12.000g at 4°C for 10 min and then subjected to oxidation in acid or base, by a method modified from Fukushima and Nixon (21). To 100μl supernatant, 20μl containing 0.5M HCl and 0.05M iodine were added for acidic or 20μl 0.5M NaOH plus 0.05M iodine for basic oxidation. After incubation for 1 hour in the dark at room temperature, 20μl 1M HCl was added to the basic oxidation only. Samples were then centrifuged for 2 min at 12.000 g and 4°C and 20μl 0.1M ascorbic acid were added for the reduction of excess iodine. Biopterin concentrations were determined by HPLC using 10μl injection volume, a Nucleosil 10 SA column (250mm long, 4mm i.d., Macherey-Nagl, Düren, Germany), eluted with 1.5ml/min 50mM potassium phosphate buffer, pH 3.0 and fluorescence detection (excitation 350nm, emission 440nm). H4B concentrations were calculated as difference of results from oxidation in acid and base, respectively.

Statistics

Results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed with SPSS 15.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 4 (GraphPad Software, La Jolla, CA, USA). When two groups were compared, a 2-tailed Student’s t-test was utilized. The Spearman rank coefficient correlation test was used to examine the relationship between microcirculatory deficits and parenchymal damage. Survival and endocrine graft function analyses were performed using the Kaplan-Meier curve and groups were compared with the log rank test. A p value of <0.05 was considered to be of statistical significance.

Results

Graft microcirculation

After 16 hours of prolonged CIT IVFM showed severe microcirculatory damage in all untreated pancreatic grafts as compared to non-transplanted controls. The various H4B treatment regimens resulted in a substantial amelioration of microcirculatory damage and preservation of the capillary net, however, with different efficacy according to the route and time of administration (Fig. 1).

Figure 1. Intravital confocal fluorescence microscopy images.

Figure 1

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(a) Severe microcirculatory impairment after 16h CIT (untreated, group I). Significant amelioration of capillary graft perfusion in all three treatment regimens: (b) H4B–enriched HTK, group II; (c) recipient pre-treatment, group III; (d) donor pre-treatment, group IV. (e) Physiological capillary net of the pancreas (non-transplanted controls, group V). Observation area: 0.00159cm2 (454μm × 349μm).

Untreated animals (group I) displayed a mean FCD of 225.7 ± 11.1 cm−1. This value was significantly lower as compared to the mean FCD of the different treatment groups (group II: 298.3 ± 15.0 cm−1, group III: 374.8 ± 8.1 cm−1, group IV: 443.3 ± 12.5 cm−1; p=0.005, p=5 × 10−6, p=10−6, respectively). However, most significant improvements were seen with H4B donor pre-treatment (group IV). In this group FCD levels reached values that were comparable and statistically not significantly different from non-transplanted controls (469.3 ± 4.9 cm−1, p=0.09) (Fig. 2a).

Figure 2. Quantification of microvascular perfusion.

Figure 2

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(a) Functional capillary density (FCD), defined as the length of all blood cell perfused nutritive capillaries per observation area. Data are presented as mean ± SEM.*p=0.005; **p=5 × 10−6; ***p=10−6; untreated (group I) vs. non-transplanted controls (group V), p=4 × 10−8; H4B–enriched HTK (group II) vs. recipient pre-treatment (group III), p=0.002; H4B–enriched HTK (group II) vs. donor pre-treatment (group IV), p=0.00008; recipient pre-treatment (group III) vs. donor pre-treatment (group IV), p=0.002.

(b) Capillary diameter (CD), defined as the mean value of the three largest capillaries per observation area. Data are presented as mean ± SEM. #p=0.05; ##p=0.007; ###p=0.009; untreated (group I) vs. non-transplanted controls (group V); all other comparisons, p=not significant.

Results of mean CD followed similar patterns. CD values were significantly lower in group I (5.3 ± 0.2 μm) compared to the three different treated groups (group II: 7.2 ± 0.2 μm, group III: 6.8 ± 0.2 μm, group IV: 7.4 ± 0.2 μm; p=0.007, p=0.05 and p=0.0009, respectively; Fig. 2b). Again, CD levels in group IV were statistically not significantly different from non-transplanted controls (7.7 ± 0.2 μm; p=0.09).

Graft histopathology

According to the Schmidt score untreated animals (group I) showed severe parenchymal damage (Fig. 3) following prolonged CIT whereas in non-transplanted controls (group V) only mild graft edema was evident. Pancreatic grafts perfused with H4B-supplemented HTK (group II) showed a significant decrease in interstitial edema, acinar, hemorrhagic and fat necrosis as compared to group I (p=0.0001, p=0.04 and p=0.02, respectively), while treatment of the recipient animals prior to reperfusion (group III) could only improve edema formation (p=0.0001). In contrast, donor pre-treatment with H4B in group IV showed substantial amelioration of all histopathologic parameters but inflammatory infiltration, differing significantly from untreated animals (p=0.0001, p=0.001 and p=0.001, respectively) as well as from the other two treatment regimens (acinar necrosis and haemorrhagic/fat necrosis, group II vs group IV: p=0.01 and p=0.05, respectively; group III vs group IV: p=0.002 and p=0.003, respectively) (Fig. 3f). In addition, a significant inverse correlation was observed between histopathologically diagnosed parenchymal damage and microcirculatory parameters (Spearman r=−0.86, p=0.0001; Fig. 3g).

Figure 3. Graft histopathology (H&E) and Schmidt score.

Figure 3

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(a) Severe parenchymal damage after 16h CIT. (b – d) All three different treatment regimens show protective effects on graft parenchyma. (e) Non-transplanted controls with slight edema. (f) Tab graph reporting the semiquantitative histopathological Schmidt score including interstitial edema, acinar, hemorrhagic and fat necroses and inflammatory infiltrates. Data are presented as mean ± SEM. *p<0.05; **p=0.02; ***p<0.003; ****p=0.0001. (g) Scatter chart showing the significant inverse correlation between Schmidt score and FCD.

Peroxynitrite and nitrotyrosine formation

Semiquantitative IHC-scoring revealed increased intragraft peroxynitrite (ONOO−) formation after prolonged cold ischemia (group I) compared to non-transplanted controls (2 ± 0 vs 6 ± 0; p=not applicable). Compared to untreated animals all H4B treatment regimens lead to a substantial decrease of ONOO− formation (group II: 4.5 ± 0.5, group III: 4.4 ± 0.4, group IV: 1.3 ± 0.25; p=0.01, p=0.004, p=10−7, respectively; Fig. 4a-4e). Grafts with H4B–supplemented perfusion solution HTK (group II) and grafts of H4B treated recipients (group III) thereby displayed similar semiquantitative IHC – scores (p=0.88). Again, donor pre-treatment (group IV) resulted in statistically significantly stronger abrogation of ONOO− - production as compared to the two other treatment options (group II vs group IV: p=0.001; group III vs group IV: p=0.0004; Fig. 4f). Peroxynitrite formation was also found to significantly inversely correlate with microcirculatory parameters (Spearman r=−0.87, p=0.0001; Fig. 4g)

Figure 4. Graft nitrotyrosine immunohistochemistry (IHC).

Figure 4

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(a) Strong staining intensity after 16h CIT. (b – d) All three different treatment regiments show diminished nitrotyrosine staining. (e) Non-transplanted controls. (f) Tab graph reporting the semiquantitative score as product of positive stained cells and staining intensity. Data are presented as mean ± SEM. *p=0.01; **p=0.004; ***p=10−7; #p=0.001; ##p=0.0004. (g) Scatter chart showing the significant inverse correlation between the nitrotyrosine staining score and the FCD.

These data were further confirmed and substantiated by nitrotyrosine Western blots which also showed almost an abrogation of ONOO− if donors were pretreated with H4B as compared to untreated grafts (p=0.002; Fig.6).

Figure 6. Nitrotyrosine Western Blot.

Figure 6

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a) One representative Western Blot of each group A – G is shown (n=9). b) Compared to controls donor pre-treatment (group IV) shows significantly decreased intragraft nitrotyrosine formation as early as 2h after organ reperfusion (p=0.002). However, no significant differences are found immediately after organ retrieval as well as prior to reperfusion. Data are presented normalized to α-actin and as mean ± SEM. A: graft immediately after organ procurement with H4B pre-treatment; B: non-transplanted control; C: graft prior to reperfusion without H4B pre-treatment; D: graft prior to reperfusion with H4B pre-treatment; E: graft 2h following reperfusion without H4B pre-treatment; F: graft 2h following reperfusion with H4B pre-treatment; G: graft immediately after organ procurement without H4B pre-treatment.

Intragraft H4B levels

Both, total biopterin and absolute H4B intragraft tissue levels did not show any significant differences between untreated and non-transplanted animals (p=0.3 and p=0.4, respectively). In contrast, analysis of the ratio between H4B and its oxidized form revealed a statistically significant difference (group I: 96.3 ± 0.8 vs group II: 86.2 ± 3.7; p=0.04). At 2h after reperfusion only group IV showed a tendency towards a higher intragraft ratio as compared to untreated animals (94.3 ± 0.8; p=0.07). However, looking at additional time points, both, immediately following organ procurement (96.4 ± 0.5) as well as at the time of reperfusion (94.5 ± 0.2), in group IV the ratio was significantly higher compared to non treated animals (p=0.02 and p=0.04, respectively; Fig. 5).

Figure 5. Intragraft ratio H4B:oxH4B (oxidized H4B).

Figure 5

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Tab graph displaying the percentage of non- oxidized H4B per total biopterin in pancreatic grafts. Data are presented as mean ± SEM. *p=0.04; #p=0.02.

Survival and endocrine function

Subsequently, recipient survival and endocrine graft function were analyzed in the most potent H4B donor pre-treatment group (IV) and compared with untreated animals. While untreated animals died after prolonged CIT within 48h to72 h losing up to 13% of their preoperative body weight, 4 animals out of 5 of the H4B donor pre-treatment group survived long term (day 100 p.o.). One animal in this group died on day 26 p.o. without a detectable reason (Fig 7a).

Figure 7.

Figure 7

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(a) Survival analysis. Kaplan-Meier curve comparing non-transplanted controls (group I) and donor pre-treatment group (IV). (b) Endocrine graft function. Line chart showing glycemia blood levels in group I and group IV. Data are presented as mean ± SEM.

Daily blood glucose measurements in streptozotocin-induced diabetic mice showed that no animal of the untreated group reached normoglycemia, whereas all animals in group IV became normoglycemic within 24h after transplantation (p=0.0002; Fig 7b).

Discussion

Recently we were able to show in a mouse model, that IRI following pancreas transplantation is associated with intragraft H4B depletion. The significant amelioration of the observed parenchymal damage through H4B substitution clearly evidenced its pathophysiologic importance. However, recipient animals in this study were treated over a 24h time period prior to transplantation, which cannot be translated into clinical practice with deceased donors. Therefore, in this study we examined three, clinically feasible H4B treatment regimens.

We opted either for H4B-supplementation to the perfusion solution HTK®, for a single H4B application to the recipient prior to organ reperfusion and for a single dose H4B treatment of the donor prior to organ retrieval. Major findings of this study were that (1) donor pre-treatment with H4B almost completely abrogated microvascular disorders and IRI-related early parenchymal damage, (2) the depletion of H4B is already initiated during organ retrieval, confirming the importance of donor pre-treatment, and most importantly (3) the efficiency of abrogating IRI by a single dose application prior to organ procurement might open new perspectives for its possible clinical use.

NO is a free radical produced by three different NOS isoforms: one inducible (iNOS) and two constitutively active (neuronal, nNOS; endothelial, eNOS). NO produced in endothelial cells by eNOS is responsible for vascular homeostasis, regulating vascular tone and permeability, and inhibiting platelet and leukocyte aggregation to endothelial surface (10, 22, 23).

There is a very fragile balance for endothelial NO production and H4B seems to play herein a key role. H4B is both an essential co-factor of NOS but also a strong antioxidant. If H4B is limited, the electron transfer needed for L-arginine oxidation becomes uncoupled resulting in increased superoxide production (24). Furthermore, superoxide itself reacts with NO forming ONOO−, which in turn oxidizes H4B further reducing its bioavailability (25).

H4B depletion and eNOS derived superoxide generation has been associated with various cardiovascular diseases (15), (16), (26, 27) and cigarette smoking (28). H4B supplementation led to substantial amelioration of pathophysiologic changes in these vascular disorders. There is increasing evidence, that H4B-depletion represents also a crucial factor in IRI following solid organ transplantation (29, 30).

IVFM is currently the gold standard to analyze microcirculatory changes. It is widely used in animal models (31) and there are also some efforts for clinical in vivo image-acquisition (32, 33). In our experimental setting FCD and CD were dramatically impaired, reflecting the deleterious effect of IRI. This is in line with observations of other groups which found a strong correlation between microcirculatory disorders and IRI (33), (34). Even though all three H4B treatment regimens were able to significantly ameliorate both IVFM-parameters, donor pre-treatment with H4B was the only regimen leading to an almost complete abrogation of microcirculatory deficits.

Intragraft parenchymal damage represents another hallmark of IRI following pancreas transplantation. The pancreatitis score published by Schmidt et al. was applied (20). This score quantifies tissue damage in four different categories (interstitial edema, acinar necrosis, hemorrhage/fat necroses, inflammatory infiltrates) with a score ranging from 0 to 4. Prolonged CIT led to a severe impairment of the pancreatic tissue in the non-treated group. Again, substantial improvements in all H4B-treated groups were observed, but the best results were obtained following donor pre-treatment. Marked differences were seen essentially in the categories acinar necrosis and hemorrhage/fat necrosis. In untreated animals these two categories testified for severe graft damage (mean score 3). Only H4B pre-treatment of the donor showed mild signs of necrosis (mean score below 1), whereas the other two treatment regimes resulted in more prominent signs of necrosis (mean scores around 2). Parenchymal edema observed in non-transplanted controls can be explained by the trauma caused by the retrieval process and by the observed higher risk for parenchymal edema in HTK-perfused pancreatic grafts (35-37). The low scores regarding inflammatory infiltrates have to be attributed to the short reperfusion period of only 2h.

The reaction of NO with oxygen radicals like superoxide results in formation of ONOO−. This potent oxidant leads to tissue injury by DNA cleavage, by lipid peroxidation and by depletion of antioxidant storages, e.g. H4B (38, 39). ONOO−-induced nitrotyrosine formation is an established marker for oxidative tissue damage (40), (41). Strong immunostaining was observed in untreated pancreatic grafts. In accordance to histopathological findings pre-treatment of the donor with H4B not only led to a significant decrease of nitrotyrosine staining but showed even significantly lower semi-quantitative scores compared to the other treatment regimens. Interestingly, non-transplanted organs showed similar scores to grafts with H4B pre-treatment. This may be attributed to surgical trauma endorsing the importance of H4B substitution already prior to organ retrieval.

Intragraft H4B tissue levels were determined in light of the H4B depletion observed after IRI in different experimental transplant models. A recent study highlighted the importance of the ratio between H4B and its oxidized form rather than the H4B total amount as reliable value for tissue level determination (42). Non-transplanted and untreated animals differed significantly from each other in this ratio. As expected, group II and group III did not differ substantially from the untreated group. Surprisingly, donor pre-treatment with H4B showed only a trend towards significance compared to untreated animals. Assuming, that this could be due to high co–factor consumption during organ retrieval, implantation and reperfusion, we additionally measured H4B intragraft tissue levels in this group immediately after organ procurement and before reperfusion of the graft. In fact, significantly different ratios were observed at these two time points, suggesting a high consumption of H4B not only during the first 2h following reperfusion but already during organ retrieval. This rather rapid elimination of H4B (43) supports the hypothesis that higher H4B tissue levels during the early period of graft damage should be sufficient to prevent amplification and progression of an IRI-associated inflammatory cascade.

Finally, the most promising regimen, H4B given to the donor, was chosen for further analysis. Western Blots confirmed significant abrogation of ONOO− formation. While severe graft pancreatitis in untreated animals was not compatible with prolonged recipient survival (≤72h), donor pre-treatment with H4B led to survival times of up to 100 days post transplantation. One animal died at day 26 of unknown reason. These observations correlate well with the reported high risk for graft loss associated with severe graft pancreatitis in human pancreas transplantation (44, 45).

Similarly, the protective effect of H4B donor pre-treatment was confirmed by analysis of endocrine graft function. Whereas all animals after H4B donor pre-treatment showed normoglycemia already at 24h after transplantation, untreated animals never reached a normoglycemic state.

The most prominent finding in this study is the superiority of the donor pretreatment group in protecting pancreatic grafts from IRI throughout all performed analyses compared to the other treatment regimens. This points the focus on recent efforts to improve donor management in order to increase the use of marginal or extended criteria donors (46). Donor pre-treatment may serve as an important option to reduce unspecific inflammatory injuries in transplanted organs due to the retrieval procedure, prolonged cold and warm ischemia time as well as reperfusion. In this regard the efficacy of agents like dopamine (47) and amiodarone (48) has already been observed. In a prospective randomized controlled clinical trial treatment of the donor with methylprednisolone significantly reduced inflammation and IRI leading to favorable post transplant liver graft function (49). In view of the danger hypothesis and the synergistic effects between nonspecific injury and alloreactive recipient responses (50) our results represent a promising additional tool to optimize solid organ quality and hence improving graft outcome.

In summary, we were able to demonstrate in a mouse model that a single dose of the essential NOS – co-factor H4B effectively protects the pancreatic graft after prolonged CIT abrogating ONOO− formation. Among the three clinically applicable treatment regimens tested in this study only donor pre-treatment with H4B was able to abrogate deleterious parenchymal damage, leading to improved graft function and recipient survival.

H4B donor pre-treatment might therefore be a promising therapeutic strategy to ameliorate or prevent IRI following human pancreas transplantation.

Acknowledgements

We are indebted to Nina Madl and Petra Loitzl for expert technical assistance.

This study has been supported by grant 22289 of the “Fonds zur Förderung der wissenschaftlichen Forschung” (FWF) and by grant #185 of the ,,Medizinischer Forschungsfonds (MFF) Tirol“.

Footnotes

[Author manuscript. The definitive version is available at www.onlinelibrary.wiley.com.]

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