Dirk Troitzsch

Protein S-100β in Brain and Serum after Deep Hypothermic Circulatory Arrest in Rabbits: Relationship to Perivascular Astrocytic Swelling

Abstract The aim of this study was to evaluate the relationship between the kinetic patterns of the protein S-100β an astroglial cell marker, and its immunohistochemical expression in the brain in rabbits that underwent cardiopulmonary bypass with deep hypothermic circulatory arrest. Fourteen New Zealand rabbits (weight, 3.1±0.25 kg) were anaesthetised, intubated and mechanically ventilated. Four animals were not connected to the cardiopulmonary bypass and served as controls. Ten animals were perfused according to a uniform protocol. After systemic cooling, deep hypothermic circulatory arrest was induced for 60 minutes. After reperfusion and rewarming, the animals were weaned from bypass and sacrificed. In the brain, astrocyte reactivity for S-100β was evaluated immunocytochemically (DPC® Immustain) and the serum concentrations of S-100β were analysed using a commercially available immunoluminometric kit (Byk-Sangtec®, Dietzenbach, Germany). In all experimental animals a significant increase of the serum concentration of the protein S-100β was found immediately after reperfusion and the termination of cardiopulmonary bypass. In comparison with the control animals, increased staining of S-100β was found in the astroglial cells and swollen astrocytic end-feet in the perivascular regions. There were fewer signs of neuronal cell injury of neurones in the hippocampus structure. In conclusion, astrocytic activation and S-100β over-expression seems to precede the neurodegeneration following deep hypothermic circulatory arrest. The marked perivascular cell swelling may support the assumption of reperfusion injury of the astroglial cell complex that forms the blood-brain barrier, which may be indicative of the source of the released S-100β into the blood stream.

Large-dose pretreatment with methylprednisolone fails to attenuate neuronal injury after deep hypothermic circulatory arrest in a neonatal piglet model.

Conflicting results have been reported with regard to the neuroprotective effects of steroid treatment with cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). We evaluated the mode and severity of neuronal cell injury in neonatal piglets after prolonged DHCA and the possible neuroprotective effect of systemic pretreatment (>6 h before surgery) with large-dose methylprednisolone (MP). Nineteen neonatal piglets (age, <10 days; weight, 2.1 ± 0.5 kg) were randomly assigned to 2 groups: 7 animals were pretreated with large-dose systemic MP (30 mg/kg) 24 h before surgery, and 12 animals without pharmacological pretreatment (saline) served as control groups. All animals were connected to full-flow CPB with cooling to 15°C and 120 min of DHCA. After rewarming to 38.5°C with CPB, animals were weaned from CPB and survived 6 h before they were killed, and the brain was prepared for light and electron microscopy, immunohistochemistry, and TUNEL-staining. Quantitative histological studies were performed in hippocampus, cortex, cerebellum, and caudate nucleus. Systemic pretreatment with large-dose MP lead to persistent hyperglycemia but no significant changes of cerebral perfusion. Necrotic and apoptotic neuronal cell death were detected in all analyzed brain regions after 120 min of DHCA. In comparison to the control group, large-dose pretreatment with systemic MP lead to an increase of necrotic neuronal cell death and induced significant neuronal apoptosis in the dentate gyrus of the hippocampus (P = 0.001). In conclusion, systemic pretreatment with large-dose MP fails to attenuate neuronal cell injury after prolonged DHCA and induces regional neuronal apoptosis in the dentate gyrus.

Improved myocardial preservation with short hyperthermia prior to cold cardioplegic ischemia in immature rabbit hearts

OBJECTIVE Recent observations have been shown that the induction and accumulation of heat shock proteins (HSPs) by short exposure to nonlethal whole-body hyperthermia with normothermic recovery are closely associated with transient resistance to subsequent ischemia-reperfusion challanges. Here, this study was performed to investigate whether a shortly heat shock pretreatment affects the left ventricular (LV) function after cold cardioplegic ischemia in reperfused neonatal rabbit hearts. METHODS Hearts from neonatal New Zealand White rabbits were isolated perfused (working heart preparation) and exposed to 2 h of cold cardioplegic ischemia followed by reperfusion for 60 min. To induce the heat shock response neonatal rabbits (n=5, HT-group) were subjected to whole-body hyperthermia at 42.0-42.5 degrees C for 15 min, followed by a normothermic recovery period of 60 min, before harvesting and the onset of global hypothermic cardioplegic arrest. Another set of hearts (n=5, control group) without a heat treatment underwent a similar perfusion and ischemia protocol served as control. The postischemic recovery was assessed by measuring several parameters of LV function. LV biopsies from all control and heat treated animals were taken before ischemia and at the end of reperfusion to examine myocardial HSP levels by Western blot analysis. RESULTS At 60 min of reperfusion the HT-group showed significant better recovery of ventricular function such as LV developed pressure (DP) (74.6+/-10 vs. 52.1+/-8.5%, P<0.05), LV positive dP/dt (910+/-170 vs. 530+/-58 mmHg/s, P<0.01) and LV end-diastolic pressure (LVEDP) (8+/-2 vs. 18.4+/-5 mmHg, P<0.05) than control. Myocardial oxygen consumption (MVO(2)) was significantly higher in the HT-group compared with control (0.054+/-0.006 vs. 0.041+/-0.002 ml/g per min, P<0.05). Significant postreperfusion lower level in lactate production was observed in the HT-group (0.83+/-0.11 vs. 1.67+/-0.8 mmol/l, P<0.05). Also, the recovery of hemodynamic parameters such as aortic flow, coronary flow and cardiac output was significantly superior (P<0.05) in the HT-group. Furthermore, high expression of HSP72(+)/73(+) were detected in the myocardial tissue samples of heat-treated rabbits by immunoblotting, appearing even at 60 min of normothermic recovery after heat stress. CONCLUSIONS These data in the immature rabbit heart indicate that previous shortly heat treatment with high level expression of heat shock proteins (HSP72(+)/73(+)) before hypothermic cardioplegic ischemia provides transient tolerance against myocardial injury and could be an improvement for the postischemic functional recovery of neonatal hearts.

Importance of Real-Time Tissue Oximetry: Relationship to Muscle Oxygenation and Tissue Viability

BACKGROUND There is currently no efficient and reliable clinical means of assessing the degree of ischemia- and reperfusion-associated damage in microvascular transplants. The objective was to study correlation of tissue oxygen tension measurements with tissue oxygen saturation, cytochrome oxidase redox state, and tissue viability. MATERIALS AND METHODS Latissimus dorsi muscle was dissected and mobilized in New Zealand white rabbits (n = 30, 2.5 ± 0.5 kg). Muscles were exposed to warm ischemia in two groups with either 4 or 6 h, followed by reperfusion. Tissue PO(2) was measured with a miniature intramuscular oxygen sensor (Licox microprobe; Integra Neurosciences, Ratingen, Germany) all along with tissue hemoglobin saturation (rSO(2)) and cytochrome oxidase aa3 redox state (CytOx) by in vivo near-infrared spectroscopy. Linear correlation was performed between tissue PO(2) and rSO(2), CytOx and tissue viability. RESULTS After ischemia and reperfusion, tissue PO(2) and CytOx recovery was significantly decreased in both groups compared with control (4 h: P < 0.05; 6 h: P < 0.01). Significant correlations between changes in tissue PO(2) and rSO(2) (r = 0.92; P < 0.01), CytOx (r = 0.90; P < 0.01), wet-to-dry ratio (r = -0.97; P < 0.01), and mitochondrial viability index (r = 0.97; P < 0.01) were found. CONCLUSIONS Tissue oxygen tension measured with microprobes correlated closely with tissue oxygenation, cellular oxygen utilization, and the extent of ischemia reperfusion injury.

Effects of cyclosporine pretreatment on tissue oxygen levels and cytochrome oxidase in skeletal muscle ischemia and reperfusion.

ABSTRACT We hypothesized that pretreatment with single-dose cyclosporine (CsA) prevents alterations and improves tissue oxygen and mitochondrial cytochrome oxidase redox (CytOx) state in skeletal muscle ischemia and reperfusion-reoxygenation (I/R). Latissimus dorsi muscle was prepared and mobilized in New Zealand white rabbits. Ischemia was induced for 4 h, followed by 2 h of reperfusion. The animals were randomized to receive a 60-mg/kg intravenous bolus of CsA (CsA group, n = 10) or physiologic saline (control, n = 10) at 10 min before ischemia onset. Muscle tissue oxygen tension (PtO2) and mitochondrial CytOx were measured during I/R simultaneously. High-energy phosphate (HEP) levels were determined using high-field 31P magnetic resonance spectroscopy. Mitochondrial viability index and wet-to-dry ratio were used to assess the tissue viability between groups. Decreases in tissue oxygen levels and CytOx were slower during ischemia in the CsA group in comparison to control group, also the loss of phosphocreatine and adenosine triphosphate depletion. After ischemia, recovery of tissue oxygen, mitochondrial CytOx, and HEP was delayed in controls. Tissue PtO2 in the CsA group (P < 0.05) was significantly higher compared with that in the control group after I/R. Mitochondrial CytOx was also improved in the CsA group (P < 0.01 vs. control). Muscle HEP levels (phosphocreatine, adenosine triphosphate) were significantly preserved in the CsA group versus the control group (P < 0.01, P < 0.05). Mitochondrial viability index and wet-to-dry ratio confirmed significantly preserved tissue and lower edema formation in the CsA group. The pretreatment with single-dose CsA prevents alterations and improves tissue oxygenation and mitochondrial oxidation in skeletal muscle I/R.

Ischemic Preconditioning Results in an Atp-dependent Inhibition of Cytochrome C Oxidase

Purpose This study addresses the effect of short myocardial ischemia on inhibitory effect of ATP for mitochondrial cytochrome c oxidase (CytOx) activity in myocardium and subsequent hemodynamic alterations. The activity of CytOx is inhibited by ATP (primary substrate control). This additional mechanism was proposed to be switched off at higher mitochondrial membrane potential values in case of stress. The ATP-dependent allosteric enzyme inhibition (second respiratory control) is suggested to reduce the formation of reactive oxygen species and thus is pivotal for cytoprotection. This report addresses the possible involvement of this mechanism in case of myocardial preconditioning. Methods Rat hearts were perfused in a Langendorff system (n = 5 each group). The first two groups underwent short recurrent ischemic periods (three times 5 min) and subsequent high or low reperfusion for 40 min. Besides four control groups, hearts were exposed to an ischemia of 15 min and high flow reperfused for 30 min, in addition. Hemodynamic data were evaluated in parallel. Mitochondria were separated for the polarographic respiration measurements in the presence of ADP or ATP, respectively. Phosphorylation patterns of the CytOx subunits were studied by immunoblotting with P-Ser, P-Thr, and P-Tyr antibodies. Results Short recurrent episodes of ischemia result in an ATP-dependent inhibition of CytOx. Electrophoretic analysis and blotting techniques reveal different phosphorylation patterns of the enzyme. Frequent short-lasting ischemic impacts and subsequent increased coronary flow seem to be essential for this effect. Conclusion The procedure of preconditioning is likely to be dependent on the mechanism of ATP-dependent inhibition of CytOx activity.

Microvascular tissue oxygenation and oxidative metabolism changes in the pedicled latissimus dorsi muscle during graded hypoxia: correlation between near infrared and 31P nuclear magnetic resonance spectroscopy.

BACKGROUND In this study, the microvascular tissue oxygenation and oxidative muscle metabolism during graded hypoxia and reoxygenation were examined in a rabbit model by near-infrared (NIR) spectroscopy and correlated with high-energy phosphates measured by (31)P nuclear magnetic resonance (NMR) spectroscopy. MATERIALS AND METHODS Graded hypoxia was performed in a New Zealand rabbit model (n = 20, 2.0 ± 0.4 kg) by a stepwise reduction of the fraction of inspired oxygen (FiO(2)) from 0.3 to 0.05 (intervention group versus control group). Recovery and reoxygenation were achieved using FiO(2) of 0.3. A noninvasive NIR spectroscopy sensor and NMR probe was positioned on the surface of the prepared pedicled latissimus dorsi muscle. Microvascular tissue oxygenation (oxyhemoglobin, HbO(2); deoxyhemoglobin, HHb) and redox state of cytochrome oxidase (CytOx) were measured by NIR spectroscopy and correlated with standard values of oxidative muscle metabolism (phosphocreatine, PCr; adenosine triphosphate, ATP) measured by time-resolved (31)P NMR spectroscopy (4.7T). RESULTS Significant correlation was found between PCr and HbO(2) (r = 0.85, P < 0.001) and HHb (r = -0.75, P < 0.001). β-ATP levels correlated significantly with CytOx (r = 0.87, P < 0.001). CONCLUSIONS The data suggest that changes in high-energy phosphates (PCr- and ATP-levels) correlate closely with microvascular tissue oxygenation (HbO(2), HHb, CytOx) measured by NIR spectroscopy.

Epicardial measurement of alterations in extracellular pH and electrolytes during ischemia and reperfusion in cardiac surgery / Epikardiale Messung von pH-Wert- und Elektrolyt-Veränderungen während Ischämie und Reperfusion in der Herzchirurgie

Abstract Simultaneous measurements of extracellular pH, potassium (K+), and calcium (Ca2+) activity might be indicative of myocardium vitality or ischemia. Ten consecutive patients undergoing elective coronary artery bypass grafting were studied. Epicardial extracellular pH, potassium, and calcium were measured by a miniaturized disposable multi-sensor probe. Blood gases and electrolytes were derived with measurements of arterial and mixed venous blood samples at intervals during surgery. The mean epicardial baseline levels for pH in all patients were 8.04±0.22 arbitrary units (AU) for the right ventricle (RV) and 8.03±0.21 AU for the left ventricle (LV); for Ca2+ 0.23±0.07 mmol/l (RV) and 0.20±0.10 mmol/l (LV); and for K+ 4.54±1.51 mmol/l (RV) and 4.38±0.57 mmol/l (LV). Before ischemia, epicardial pH was moderately (p<0.05), and K+, and Ca2+ were closely correlated (p<0.001) with blood values. During reperfusion, epicardial measurements were weakly correlated (p<0.001) with blood values for pH, venous K+ and Ca2+, but moderately correlated with arterial K+ and Ca2+ (p<0.01). The measurements indicated intraoperative episodes of ischemia and reperfusion with reproducible trends of extracellular pH, K+, and Ca2+, which results in electrolyte patterns applicable for detecting inadequate myocardial protection during cardiac surgery in patients. Zusammenfassung Die gleichzeitige Messung des extrazellulären pH und der Kalium- oder Calciumaktivitäten kann als Indikator für die Vitalität des Myokards bzw. einer Ischämie angesehen werden. Bei 10 Patienten mit der Indikationsstellung zur aortokoronaren Bypass-Operation wurden diese Parameter intraoperativ mit einem miniaturisiertem Multi-Sensor-Messkopf erfasst. Entsprechend festgelegten Intervallen erfolgten diese Messungen zusammen mit entsprechenden Blutgasanalysen des arteriellen und gemischt-venösen Blutes. Die Ausgangswerte für den pH lagen bei 8,04±0,22 AU für den rechten Ventrikel (RV) und 8,03±0,21 AU für den linken Ventrikel (LV). Die Elektrolytaktivitäten betrugen für Ca2+ 0,23±0,07 mmol/l (RV) und 0,20±0,10 mmol/l (LV) bzw. für K+ 4,54±1,51 mmol/l (RV) und 4,38±0,57 mmol/l (LV). Vor dem Zeitpunkt einer Ischämie zeigte sich eine geringe (p<0,05) Korrelation zwischem dem epikardialen pH und den Blutwerten. Die Elektrolyte wiesen eine hohe Korrelation (p<0,001) auf. Während der Reperfusion wiesen die epikardialen Messungen nur schwache Korrelationen zu den venösen Blutwerten auf. Andererseits fand sich eine Korrelation mit den arteriellen Elektrolytwerten (p<0,01). Die Messungen gestatteten eine intraoperative Erfassung der myokardialen Ischämie und Reperfusion durch reproduzierbare Verläufe des extrazellulären pH und der Elektrolytaktivitäten, so dass dadurch der Nachweis einer inadäquaten Myokardprotektion während eines herzchirurgischen Eingriffs ermöglicht wird.

Myokardialer Ischämienachweis durch epikardiale Detektion von Kaliumionenaktivitäten / Diagnosis of Myocardial Ischaemia by Epicardial Detection of Potassium Ion Activity

Die frühzeitige Erkennung einer Myokardischämie ist in der kardiologischen und herzchirurgischen Intensivmedizin ein zentrales Problem. Ein neuer Ansatz dafür besteht in der Verwendung kaliumionenselektiver Elektroden, die, direkt epikardial implantiert, durch einen Anstieg der Kaliumionenaktivität auf dem Kammermyokard eine Hypoxie anzeigen. Nach detaillierter Sondencharkterisierung sollte in einem Tiermodell die Anwendung dieses Detektionssystems überprüft werden. Durch kontrollierte, respiratorisch induzierte Hypoxie wurde ein Anstieg der epikardialen Kaliumionenaktivität induziert (p < 0,01). Die parallel ausgeführten Blutgasanalysen belegten einen reduzierten arteriellen p02, jedoch keine Azidose. Die hämodynamischen Daten deuten auf eine hypoxisch bedingte Kreislaufdepression. Die histologischen Untersuchungen des ionenselektiv-detektierten Myokards wiesen eine typische Lymphozyteninfiltration auf. Eine Gewebeschädigung konnte nicht nachgewiesen werden. Elektronenmikroskopisch wurde eine Cristolyse in den Mitochondrien als Zeichen einer frühen Myokardschädigung nachgewiesen. Dadurch konnte die Empfindlichkeit des Elektrodensystems unter Beweis gestellt werden. Damit wird die Notwendigkeit zur Überprüfung dieses Systems im klinischen Einsatz unterstrichen. Early detection of myocardial ischaemia is a central problem in cardiological and cardiosurgical intensive care. A new approach is the use of ion-selective electrodes implanted directly on the myocardium, enabling detection of increased potassium activity as an indication of general hypoxia. After a comprehensive study of the electrode parameters, an animal experiment was carried out, in which it was found that respiration-induced hypoxia resulted in an increase in epicardial potassium activity (p<0.01). Blood gas analysis performed simultaneously revealed reduced arterial p02, but no acidosis. Haemodynamic data evidenced hypoxic depression of circulatory parameters. Histological examinations of the myocardium beneath the electrodes revealed typical lymphocytic infiltration. Electron microscopy demonstrated crystolysis in the mitochondria as an early sign of hypoxia, thus confirming the sensitivity of these electrodes. This underscores the potential of ion-selective electrodes for the detection of myocardial ischaemia, and they should now be investigated in the clinical setting.

Direct measurement of myocardial oxygen tension and high energy phosphate content under varying ventilatory conditions in rabbits / Messung von myokardialem Sauerstoffpartialdruck und energiereichen Phosphaten unter verschiedenen Beatmungsbedingungen beim Kaninchen

Abstract Effective myocardial oxygen supply should not be compromised during cardiac surgery as it is essential to avoid circulatory and cardiac dysfunction. Local measurement of myocardial oxygen partial pressure (pO2) was therefore introduced into the operative monitoring of myocardial ischemia. The aim of the present study was to assess whether myocardial oxygen partial pressure correlates with the content of high energy phosphates (HEPs). Seven male rabbits were examined in parallel with measurement of myocardial pO2 by an implanted Clark electrode and 31phosphorus-NMR spectroscopy. The ventilatory management established hyperoxygenation followed by systemic hypoxia with hypercapnia for 20 min. Additionally, analysis of end-expiratory gas composition in combination with blood gas analysis was performed simultaneously, and hemodynamic parameter was recorded. Under hypoxic conditions the cardiovascular system was severely compromised, whereas the myocardial pO2 was only moderately impaired (pO2M 45.0±16.0 mm Hg). Immediately before cardiac arrest, low values of arterial and venous pO2 were found (17.6±6.0 and 12.9±6.1 mm Hg). In contrast to near normal myocardial pO2, HEP content in the myocardium was considerably reduced and inorganic phosphorus was increased. Artificial ventilation leading to systemic hypoxia and eventually circulatory arrest resulted in almost normal myocardial pO2 but severely compromised HEP content. This somewhat unexpected finding requires further clarification, but is in accordance with findings reported previously where regulatory mechanisms have been shown to play a role in the pathophysiology of severe hypoxic conditions such as those for cellular oxygen delivery and demand, P/O coupling and finally control of HEP production facilitating the interaction between respiratory chain and myoglobin oxygen transport. Zusammenfassung Für die Abwendung eines Herz-Kreislauf-Versagens ist eine ausreichende Sauerstoffversorgung des Herzens unerlässlich. Im Verlauf eines kardiochirurgischen Eingriffes ist daher gegen eine Beeinträchtigung der myokardialen Sauerstoffversorgung vorzusorgen. Für den Nachweis einer perioperativen Ischämie findet mittlerweile die direkte Messung des regionalen myokardialen Sauerstoffpartialdruckes Anwendung. In der vorliegenden Studie wurde untersucht, inwieweit diese Messung mit dem aktuellen myokardialen Gehalt an energiereichen Phosphaten korreliert. An sieben männlichen Kaninchen wurden parallel der myokardiale Sauerstoffpartialdruck über eine implantierte Clark-Elektrode und der Gehalt an energiereichen Phosphaten mittels 31P-NMR-Spektroskopie bestimmt. Entsprechend dem gewählten Beatmungsmuster erfolgten eine Hyperoxygenation und systemische Hypoxie mit begleitender Hypercapnie über ein Intervall von 20 Minuten. Begleitend wurden simultane Atemgasanalysen, Blutgasanalysen und die Aufzeichnung hämodynamischer Daten durchgeführt. Unter diesen extrem hypoxischen Bedingungen fanden sich bei deutlich eingeschränkten Kreislaufparametern nur geringfügig verminderte Sauerstoffpartialdrücke (pO2M=45,0±16,0 mm Hg) am Messpunkt. Niedrigere Sauerstoffpartialdrücke fanden sich im arteriellen und venösen Blut (17,6±6,0 and 12,9±6,1 mm Hg) unmittelbar vor dem Herzstillstand. Im Gegensatz dazu war der myokardiale Gehalt an energiereichen Phosphaten (HEP) deutlich reduziert und das anorganische Phosphat exzessiv erhöht. Diese gegensätzlichen Befunde unterstreichen die Notwendigkeit weiterer Untersuchungen. Unter deutlich eingeschränkter Beatmung kam es zum Kreislaufversagen, obwohl sich fast normale Sauerstoffpartialdrücke im Myokard zeigten. Neben der notwendigen Überprüfung der Grenzen des Messsystems müssen jedoch auch Regulationsmechanismen, die zwischen Sauerstoffangebot und -verbrauch, der Kopplung von Phosphorylierung und Oxidation sowie bei der HEP-Synthese überprüft werden, da sie die Wechselwirkung zwischen Atmungskette und Sauerstofftransport durch Myoglobin bestimmen.