Andreas Puntschart

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

BackgroundAs 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).MethodsBD 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.ResultsIn 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.ConclusionThe 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.

A 10 min “no-touch” time – is it enough in DCD? A DCD Animal Study

Summary Donation after cardiac death (DCD) is under investigation because of the lack of human donor organs. Required times of cardiac arrest vary between 75 s and 27 min 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 2 min up to 10 min; then 30 min of CPR were performed. Brain death was determined by established clinical and electrophysiological criteria. In all animals with cardiac arrest of at least 6 min, a persistent loss of brainstem reflexes and no reappearance of bioelectric brain activity occurred. Reappearance of EEG activity was found until 4.5 min of cardiac arrest and subsequent CPR. Brainstem reflexes were detectable until 5 min of cardiac arrest and subsequent CPR. According to our experiments, the suggestion of 10 min 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.

Establishing a brain-death donor model in pigs.

INTRODUCTION An animal model that imitates human conditions might be useful not only to monitor pathomechanisms of brain death and biochemical cascades but also to investigate novel strategies to ameliorate organ quality and functionality after multiorgan donation. METHODS Brain death was induced in 15 pigs by inserting a catheter into the intracranial space after trephination of the skull and augmenting intracranial pressure until brain stem herniation. Intracranial pressure was monitored continuously; after 60 minutes, brain death diagnostics were performed by a neurologist including electroencephalogram (EEG) and clinical examinations. Clinical examinations included testing of brain stem reflexes as well as apnoe testing; then intensive donor care was performed according to standard guidelines until 24 hours after confirmation of brain death. Intensive donor care was performed according to standard guidelines for 24 hours after brain death. RESULTS Sixty minutes after brain-death induction, neurological examination and EEG examination confirmed brain death. Intracranial pressure increased continuously, remaining stable after the occurrence of brain death. All 15 animals showed typical signs of brain death such as diabetes insipidus, hypertensive and hypotensive periods, as well as tachycardia. All symptoms were treated with standard medications. After 24 hours of brain death we performed successful multiorgan retrieval. DISCUSSION Brain death can be induced in a pig model by inserting a catheter after trephination of the skull. According to standard guidelines the brain-death diagnosis was established by a flat-line EEG, which occurred in all animals at 60 minutes after induction.

Magen und Duodenum

Ulkusperforationen treten zwar aufgrund des verbreiteten Einsatzes von Protonenpumpeninhibitoren seltener auf, aber die zeitverzogerte Therapie ist noch immer mit einer hohen Letalitat vergesellschaftet. Bei adaquater zeitnaher Diagnostik (< 24 h) ist eine laparoskopische Sanierung (Cave: Kontraindikationen) die Therapie der Wahl und beschleunigt den Heilungsverlauf. Die histologische Aufarbeitung des Ulkusmaterials ist obligat. Magenausgangstenosen erfordern grundsatzlich kein akutchirurgisches Vorgehen, jedoch sind eine Hospitalisierung des Patienten (Elektrolytverlust, Alkalose etc.) und eine exakte Diagnostik (haufig neoplastischer Grundprozess) notwendig. Diagnostisch ist eine Osophagogastroduodenoskopie mit Biopsie unumganglich. Therapeutisches Ziel (kurativ bzw. palliativ) ist die Wiederherstellung der Magen-Darm-Passage. Grundsatzlich ist bei beiden Erkrankungsbildern an eine Helicobacter-Eradikationstherapie zu denken.

Hyperbaric oxygenation of UW solution positively impacts on the energy state of porcine pancreatic tissue

SummaryBACKGROUND: Pancreatic islet transplantation is a promising option for the treatment of diabetic patients; xenotransplantation of porcine islet cells would be a possibility to overcome the shortage of donor organs. Usually the donor pancreas is preserved with University of Wisconsin (UW) solution. A large number of reports have shown that the two-layer method (TLM), which employs oxygenated perfluorochemical and UW solution, is superior to simple cold storage. However, the extensive use of TLM is cost intensive and there is evidence that TLM only oxygenates small parts of the organ preserved. Another possibility to increase the oxygen supply during organ preservation would be the use of hyperbaric oxygenation (HBO) which increases the oxygen tension in fluids. The aim of this study was to evaluate the effect of pre-oxygenation of different preservation solutions on organ quality in terms of high energy phosphate levels as well as the occurrence of apoptosis and the induction of heat shock proteins and nitrosative stress induced cell death in porcine pancreatic tissue. METHODS: Porcine pancreatic tissue was preserved in different preservation solutions with or without pre-oxygenation for 6 hours of cold ischemic time (CIT). Then, tissue specimens were harvested and high energy phosphate levels were determined by HPLC. Moreover, immunohistochemistry was performed in order to detect occurrence of apoptosis, heat shock protein 70 (HSP70) as well as nitrosative stress induced cell death. RESULTS: Organs stored in pre-oxygenated UW solution showed best results in terms of high energy phosphate levels; apoptotic cells per islet as well as HSP70 positivity were significantly less when compared to simple UW storage and all other organ preservation solutions with or without pre-oxygenation. CONCLUSIONS: Pre-oxygenation of UW solution is a simple and promising method to improve islet cell quality after cold organ storage. However, further in vitro experiments have to be performed in order to confirm these findings.