Oxidative stress and apoptosis in a pig model of brain death (BD) and living donation (LD).

BACKGROUND: As organ shortage is increasing, the acceptance of marginal donors increases, which might result in poor organ function and patient survival. Mostly, organ damage is caused during brain death (BD), cold ischemic time (CIT) or after reperfusion due to oxidative stress or the induction of apoptosis. The aim of this study was to study a panel of genes involved in oxidative stress and apoptosis and compare these findings with immunohistochemistry from a BD and living donation (LD) pig model and after cold ischemia time (CIT). METHODS: BD was induced in pigs; after 12 h organ retrieval was performed; heart, liver and kidney tissue specimens were collected in the BD (n = 6) and in a LD model (n = 6). PCR analysis for NFKB1, GSS, SOD2, PPAR-alpha, OXSR1, BAX, BCL2L1, and HSP 70.2 was performed and immunohistochemistry used to show apoptosis and nitrosative stress induced cell damage. RESULTS: In heart tissue of BD BAX, BCL2L1 and HSP 70.2 increased significantly after CIT. Only SOD2 was over-expressed after CIT in BD liver tissue. In kidney tissue, BCL2L1, NFKB, OXSR1, SOD2 and HSP 70.2 expression was significantly elevated in LD. Immunohistochemistry showed a significant increase in activated Caspase 3 and nitrotyrosine positive cells after CIT in BD in liver and in kidney tissue but not in heart tissue. CONCLUSION: The up-regulation of protective and apoptotic genes seems to be divergent in the different organs in the BD and LD setting; however, immunohistochemistry revealed more apoptotic and nitrotyrosine positive cells in the BD setting in liver and kidney tissue whereas in heart tissue both BD and LD showed an increase.

Energy status of pig donor organs after ischemia is independent of donor type.

BACKGROUND: Literature is controversial whether organs from living donors have a better graft function than brain dead (BD) and non-heart-beating donor organs. Success of transplantation has been correlated with high-energy phosphate (HEP) contents of the graft. METHODS: HEP contents in heart, liver, kidney, and pancreas from living, BD, and donation after cardiac death in a pig model (n=6 per donor type) were evaluated systematically. BD was induced under general anesthesia by inflating a balloon in the epidural space. Ten hours after confirmation, organs were retrieved. Cardiac arrest was induced by 9V direct current. After 10min of ventricular fibrillation without cardiac output, mechanical and medical reanimation was performed for 30min before organ retrieval. In living donors, organs were explanted immediately. Freeze-clamped biopsies were taken before perfusion with Celsior solution (heart) or University of Wisconsin solution (abdominal organs) in BD and living donors or with Histidine-Tryptophan-Ketoglutaric solution (all organs) in non-heart-beating donors, after perfusion, and after cold ischemia (4h for heart, 6h for liver and pancreas, and 12h for kidney). HEPs (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and phosphocreatine), xanthine, and hypoxanthine were measured by high-performance liquid chromatography. Energy charge and adenosine triphosphate-to-adenosine diphosphate ratio were calculated. RESULTS: After ischemia, organs from different donor types showed no difference in energy status. In all organs, a decrease of HEP and an increase in hypoxanthine contents were observed during perfusion and ischemia, irrespective of the donor type. CONCLUSION: Organs from BD or non-heart-beating donors do not differ from living donor organs in their energy status after average tolerable ischemia.

Everolimus in different combinations as maintenance immunosuppressive therapy in heart transplant recipients.

OBJECTIVES: We examined the experiences of heart transplant recipients receiving everolimus as maintenance therapy in different combinations over a long time. MATERIALS AND METHODS: Between 2004 and 2009, forty patients (29 men, 11 women; mean age, 51.6 y) were switched from a routine immunosuppressive regimen to everolimus. Indications were other (2), renal insufficiency (17), cardiac allograft vasculopathy (14), and ongoing cellular rejection (7). Combinations were either along with cyclosporine (24), mycophenolate mofetil (14), or others (2). Indications for the introduction of everolimus including safety, efficacy, different combinations of everolimus, biopsy-proven acute rejections, renal function, and infections were evaluated retrospectively. RESULTS: Five patients died, 4 of them were still on everolimus at the time of death; they died from intracerebral hemorrhage (1), embolism (1), cardiac arrest (2), and unknown (1). Everolimus was discontinued in 6 patients owing to severe adverse effects: Edema (2), gastrointestinal adverse effects (3), and dermal adverse effects (1). Mean everolimus trough levels were 5.8 µmol/L at 6 months and 4.9 at 60 months. Mean cyclosporine levels were 67.62 µmol/L at 6 months and 47.3 µmol/L at 60 months. Mean serum creatinine levels were stable (147.9 µmol/L after 60 months). Four life-threatening infections (all pneumonia) occurred but resulted in complete recovery. CONCLUSIONS: Everolimus is safe with different immunosuppressive combinations after receiving a heart transplant.

Effect of oxidative stress and endotoxin on human serum albumin in brain-dead organ donors.

Albumin, among other molecules, binds and detoxifies endotoxin in healthy people. Oxidative stress leads to protein oxidation and thus to the impaired binding properties of albumin. This property, in combination with increased gut permeability, leads to the appearance of endotoxin in the systemic circulation and to impaired organ function. We hypothesize that these processes occur in the serum of brain-dead organ donors. Endotoxin was determined with an adapted Limulus amoebocyte lysate assay. The albumin fractions and binding capacity were determined by high-performance liquid chromatography (HPLC). FlowCytomix (eBioscience, San Diego, Calif) was used to determine the cytokine levels. Carbonylated proteins (CPs) and myeloperoxidase (MPO) were measured by an enzyme-linked immunosorbent assay (ELISA). Eighty-four brain-dead organ donors were enrolled and categorized by the duration of intensive care unit (ICU) stay. The albumin-binding capacity for dansylsarcosine was reduced in brain-dead patients compared with controls. Endotoxin positivity in 16.7% of donors was associated with decreased binding capacity in donors and worse survival of recipients. The CP and MPO levels of organ donors were significantly higher than in healthy controls. The durations of ICU stay increased albumin oxidation. In addition, interleukin-6 (IL-6), IL-8, IL-10, and IL-1ß levels were increased in patients, whereas the interferon-γ (IFN-γ) levels were within the normal range. We conclude that oxidative stress and systemic endotoxemia are present in brain-dead organ donors, which might affect recipient survival. High endotoxin levels might be caused by increased gut permeability and decreased binding capacity of albumin influenced not just by higher albumin oxidation.

A 10min "no-touch" time - is it enough in DCD? A DCD animal study.

Donation after cardiac death (DCD) is under investigation because of the lack of human donor organs. Required times of cardiac arrest vary between 75s and 27min until the declaration of the patients' death worldwide. The aim of this study was to investigate brain death in pigs after different times of cardiac arrest with subsequent cardiopulmonary resuscitation (CPR) as a DCD paradigm. DCD was simulated in 20 pigs after direct electrical induction of ventricular fibrillation. The "no-touch" time varied from 2min up to 10min; then 30min of CPR were performed. Brain death was determined by established clinical and electrophysiological criteria. In all animals with cardiac arrest of at least 6min, a persistent loss of brainstem reflexes and no reappearance of bioelectric brain activity occurred. Reappearance of EEG activity was found until 4.5min of cardiac arrest and subsequent CPR. Brainstem reflexes were detectable until 5min of cardiac arrest and subsequent CPR. According to our experiments, the suggestion of 10min of cardiac arrest being equivalent to brain death exceeds the minimum time after which clinical and electrophysiological criteria of brain death are fulfilled. Therefore shorter "no-touch" times might be ethically acceptable to reduce warm ischemia time.